Polynucleotides encoding anti-chikungunya virus antibodies

ABSTRACT

This disclosure relates to compositions and methods for treating and preventing chikungunya virus infection by delivering polynucleotides encoding anti-chikungunya virus antibodies to a subject. Compositions and treatments provided herein include one or more polynucleotides having an open reading frame encoding an anti-chikungunya virus antibody heavy chain or fragment thereof and/or an anti-chikungunya virus antibody light chain or fragment thereof. Methods for preparing and using such treatments are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Appl. No.62/613,938, filed Jan. 5, 2018, and U.S. Provisional Appl. No.62/712,599, filed Jul. 31, 2018. The content of the prior applicationsare incorporated by reference herein in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbers R01AI114816, HHSN272201400018C, W31P4Q-13-1-0003, and W911NF-13-1-0417awarded by the National Institutes of Health and the Defense AdvancedResearch Projects Agency. The government has certain rights in theinvention.

BACKGROUND

Chikungunya fever is an acute febrile illness that is caused by thechikungunya virus (CHIKV), an arthropod-borne alphavirus that istransmitted primarily by the bite of an infected Aedes species mosquito.CHIKV has caused millions of cases of disease in countries around theIndian Ocean, and has spread into novel ecological niches, includingEurope and Australia. The incubation period for chikungunya fever isusually between three to seven days. Symptoms develop abruptly with highfever that can last for several days, and severe and often debilitatingpolyarthralgias. Arthritis with joint swelling can also occur. In somecases, infected individuals can develop a maculopapular rash, anddevelop non-specific symptoms, such as headache, fatigue, nausea,vomiting, conjunctivitis, and myalgias. Chikungunya fever rarely causesdeath, but patients can have prolonged symptoms for several months.

Chikungunya fever is limited generally to supportive care, whichincludes rest, fluids, antipyretics, and analgesics. Existing drugs,such as chloroquine, acyclovir, ribavirin, interferon-α, andcorticosteroids, have been tested in vitro and in limited clinicalstudies, but these treatments are not used widely. There is an unmetneed for an improved treatment for, and prevention of, chikungunya feverin view of the limited options that are available currently.

SUMMARY

The present disclosure provides compositions and methods of preventingand/or treating disease and/or symptoms caused by chikungunya virus(CHIKV), e.g., chikungunya fever, in a subject. In some embodiments, thedisclosure relates to compositions and methods used to provide passiveimmunization against CHIKV infection. In some aspects, the disclosurerelates to compositions and methods of alleviating or reducing symptomsrelated to CHIKV infection in a subject. For example, mRNA compositionsdescribed herein can be administered to a subject confirmed as havingbeen infected by CHIKV, so as to prevent the onset of symptoms oralleviate the severity of symptoms related to CHIKV infection. In somecases, the mRNA compositions described herein can be administered to asubject suspected of having been exposed to CHIKV or being infected byCHIKV, or at risk of being exposed to CHIKV, so as to prevent the onsetof disease symptoms or to reduce the severity of symptoms.

The mRNA therapeutics of the invention are particularly well-suited forthe treatment of chikungunya fever, caused by infection by CHIKV, in asubject. The technology provides for the intracellular delivery of oneor more mRNAs encoding an anti-CHIKV antibody, followed by de novosynthesis of functional anti-CHIKV antibody within target cells. Theinstant invention features the incorporation of modified nucleotideswithin therapeutic mRNAs to (1) minimize unwanted immune activation(e.g., the innate immune response associated with the in vivointroduction of foreign nucleic acids) and (2) optimize the translationefficiency of mRNA to protein. Exemplary aspects of the inventionfeature a combination of nucleotide modification to reduce the innateimmune response and sequence optimization, in particular, within theopen reading frame (ORF) of therapeutic mRNAs encoding anti-CHIKVantibody to enhance protein expression.

In further embodiments, the mRNA therapeutic technology described hereinalso features delivery of mRNAs encoding the heavy and light chains ofan anti-CHIKV antibody via a lipid nanoparticle (LNP) delivery system.The instant disclosure features ionizable lipid-based LNPs, which haveimproved properties when combined with mRNA encoding the heavy and lightchains of anti-CHIKV antibody and administered in vivo, for example,cellular uptake, intracellular transport and/or endosomal release orendosomal escape. LNP formulations described herein also demonstratereduced immunogenicity associated with the in vivo administration ofLNPs.

In certain aspects, the disclosure relates to compositions and deliveryformulations comprising a polynucleotide, e.g., a ribonucleic acid(RNA), e.g., a mRNA, encoding a heavy chain of an anti-CHIKV antibodyand/or a polynucleotide, e.g., a ribonucleic acid (RNA), e.g., a mRNA,encoding a light chain of an anti-CHIKV antibody, and methods fortreating diseases or disorders associated with CHIKV infection, e.g.,chikungunya fever, in a human subject in need thereof by administeringthe same.

The present disclosure provides a pharmaceutical composition comprisinglipid nanoparticle encapsulated mRNAs that comprise an open readingframes (ORFs) encoding a heavy chain polypeptide of an anti-CHIKVantibody and a light chain polypeptide of an anti-CHIKV antibody,wherein the composition is suitable for administration to a humansubject in need of treatment for CHIKV infection, e.g., a human subjectwith chikungunya fever.

The present disclosure further provides a pharmaceutical compositioncomprising: (a) a mRNA that comprises (i) an open reading frame (ORF)encoding a heavy chain polypeptide of an anti-CHIKV antibody, whereinthe ORF comprises at least one chemically modified nucleobase, sugar,backbone, or any combination thereof, and (ii) an untranslated region(UTR) comprising a microRNA (miRNA) binding site; (b) a mRNA thatcomprises (i) an ORF encoding a light chain polypeptide of an anti-CHIKVantibody, wherein the ORF comprises at least one chemically modifiednucleobase, sugar, backbone, or any combination thereof, and (ii) anuntranslated region (UTR) comprising a microRNA (miRNA) binding site;and (c) a delivery agent, wherein the pharmaceutical composition issuitable for administration to a human subject in need of treatment fora disease or disorder associated with CHIKV infection, e.g., chikungunyafever.

In one aspect, the disclosure features a polynucleotide comprising anmRNA comprising: (i) a 5′ UTR; (ii) an open reading frame (ORF) encodinga polypeptide comprising the heavy chain variable region of the heavychain antibody sequence of SEQ ID NO:1, wherein the ORF comprises anucleic acid sequence that is at least 80% identical to nucleotides61-426 of SEQ ID NO:2; (iii) a stop codon; and (iv) a 3′ UTR.

In some embodiments of this aspect, the nucleic acid sequence is atleast 80% identical to nucleotides 61-1416 of SEQ ID NO:2. In someembodiments, the nucleic acid sequence is at least 80% identical to SEQID NO:2. In some embodiments, the nucleic acid sequence is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to nucleotides 61-426 of SEQ ID NO:2. In someembodiments, the nucleic acid sequence is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto nucleotides 61-1416 of SEQ ID NO:2. In some embodiments, the nucleicacid sequence is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% identical to SEQ ID NO:2.

In another aspect, the disclosure features a polynucleotide comprisingan mRNA comprising: (i) a 5′ UTR; (ii) an open reading frame (ORF)encoding a polypeptide comprising the light chain variable region of thelight chain antibody sequence of SEQ ID NO:3, wherein the ORF comprisesa nucleic acid sequence that is at least 80% identical to nucleotides61-384 of SEQ ID NO:4; (iii) a stop codon; and (iv) a 3′ UTR.

In some embodiments of this aspect, the nucleic acid sequence is atleast 80% identical to nucleotides 61-705 of SEQ ID NO:4. In someembodiments, the nucleic acid sequence is at least 80% identical to SEQID NO:4. In some embodiments, the nucleic acid sequence is at least 90%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to nucleotides 61-384 of SEQ ID NO:4. In someembodiments, the nucleic acid sequence is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto nucleotides 61-705 of SEQ ID NO:4. In some embodiments, the nucleicacid sequence is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% identical to SEQ ID NO:4.

In some embodiments of the above aspects, the mRNA comprises a microRNA(miR) binding site. In some embodiments, the microRNA is expressed in animmune cell of hematopoietic lineage or a cell that expresses TLR7and/or TLR8 and secretes pro-inflammatory cytokines and/or chemokines.In some embodiments, the microRNA binding site is for a microRNAselected from miR-126, miR-142, miR-144, miR-146, miR-150, miR-155,miR-16, miR-21, miR-223, miR-24, miR-27, miR-26a, or any combinationthereof. In some embodiments, the microRNA binding site is for amicroRNA selected from miR126-3p, miR-142-3p, miR-142-5p, miR-155, orany combination thereof. In some embodiments, the microRNA binding siteis a miR-142-3p binding site. In some embodiments, the microRNA bindingsite is located in the 3′ UTR of the mRNA.

In some embodiments of the above aspects, the 5′ UTR comprises a nucleicacid sequence at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO:13. In someembodiments of the above aspects, the 3′ UTR comprises a nucleic acidsequence at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to SEQ ID NO:14.

In some embodiments of the above aspects, the mRNA comprises a 5′terminal cap. In some embodiments, the 5′ terminal cap comprises a Cap0,Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof.

In some embodiments of the above aspects, the mRNA comprises a poly-Aregion. In some embodiments, the poly-A region is at least about 10, atleast about 20, at least about 30, at least about 40, at least about 50,at least about 60, at least about 70, at least about 80, at least about90 nucleotides in length, or at least about 100 nucleotides in length.In some embodiments, the poly-A region is about 10 to about 200, about20 to about 180, about 50 to about 160, about 70 to about 140, or about80 to about 120 nucleotides in length.

In some embodiments of the above aspects, the mRNA comprises at leastone chemically modified nucleobase, sugar, backbone, or any combinationthereof. In some embodiments, the at least one chemically modifiednucleobase is selected from the group consisting of pseudouracil (ψ),N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil, 2-thiouracil (s2U),4′-thiouracil, 5-methylcytosine, 5-methyluracil, 5-methoxyuracil, andany combination thereof. In some embodiments, at least about 25%, atleast about 30%, at least about 40%, at least about 50%, at least about60%, at least about 70%, at least about 80%, at least about 90%, atleast about 95%, at least about 99%, or 100% of the uracils areN1-methylpseudouracils.

In some embodiments, the mRNA comprises the nucleic acid sequence setforth in SEQ ID NO:5. In some embodiments, the mRNA comprises thenucleic acid sequence set forth in SEQ ID NO:6.

In some embodiments, the mRNA comprises the nucleic acid sequence setforth in SEQ ID NO:5, a 5′ terminal cap comprising Cap1, and a poly-Aregion 100 nucleotides in length. In some embodiments, the mRNAcomprises the nucleic acid sequence set forth in SEQ ID NO:6, a 5′terminal cap comprising Cap1, and a poly-A region 100 nucleotides inlength. In some embodiments, all of the uracils of the polynucleotideare N1-methylpseudouracils.

In another aspect, the disclosure provides a pharmaceutical compositioncomprising a polynucleotide described herein and a delivery agent.

In another aspect, the disclosure features a pharmaceutical compositioncomprising: a first polynucleotide comprising a first mRNA comprising(i) a first 5′ UTR, (ii) a first open reading frame (ORF) encoding afirst polypeptide comprising the heavy chain variable region of theheavy chain antibody sequence of SEQ ID NO:1, wherein the first ORFcomprises a first nucleic acid sequence that is at least 80% identicalto nucleotides 61-426 of SEQ ID NO:2, (iii) a first stop codon, and (iv)a first 3′ UTR; a second polynucleotide comprising a second mRNAcomprising (i) a second 5′ UTR, (ii) a second ORF encoding a secondpolypeptide comprising the light chain variable region of the lightchain antibody sequence of SEQ ID NO:3, wherein the second ORF comprisesa second nucleic acid sequence that is at least 80% identical tonucleotides 61-384 of SEQ ID NO:4, (iii) a second stop codon, and (iv) asecond 3′ UTR; and

a delivery agent, wherein the first polypeptide when paired with thesecond polypeptide forms an anti-Chikungunya virus antibody or ananti-Chikungunya virus antibody fragment.

In some embodiments of the above aspect, the first nucleic acid sequenceis at least 80% identical to nucleotides 61-1416 of SEQ ID NO:2, and thesecond nucleic acid sequence is at least 80% identical to nucleotides61-705 of SEQ ID NO:4. In some embodiments, the first nucleic acidsequence is at least 80% identical to SEQ ID NO:2, and the secondnucleic acid sequence is at least 80% identical to SEQ ID NO:4. In someembodiments, the first nucleic acid sequence is at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to nucleotides 61-426 of SEQ ID NO:2, and the second nucleicacid sequence is at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% identical to nucleotides 61-384 ofSEQ ID NO:4. In some embodiments, the first nucleic acid sequence is atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% identical to nucleotides 61-1416 of SEQ ID NO:2, andthe second nucleic acid sequence is at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical tonucleotides 61-705 of SEQ ID NO:4. In some embodiments, the firstnucleic acid sequence is at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:2,and the second nucleic acid sequence is at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto SEQ ID NO:4.

In some embodiments of the above aspect, the first 5′ UTR and the second5′ UTR each comprise a nucleic acid sequence at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, at least 99%, or 100%identical to SEQ ID NO:13. In some embodiments, the first 3′ UTR and thesecond 3′ UTR each comprise a nucleic acid sequence at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical to SEQ ID NO:14.

In some embodiments of the above aspect, the first mRNA and the secondmRNA each comprise a 5′ terminal cap. In some embodiments, each 5′terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′methylG cap, or an analog thereof.

In some embodiments of the above aspect, the first mRNA and the secondmRNA each comprise a poly-A region. In some embodiments, each poly-Aregion is at least about 10, at least about 20, at least about 30, atleast about 40, at least about 50, at least about 60, at least about 70,at least about 80, at least about 90 nucleotides in length, or at leastabout 100 nucleotides in length. In some embodiments, each poly-A regionis about 10 to about 200, about 20 to about 180, about 50 to about 160,about 70 to about 140, or about 80 to about 120 nucleotides in length.

In some embodiments of the above aspect, the first mRNA and the secondmRNA each comprise at least one chemically modified nucleobase, sugar,backbone, or any combination thereof. In some embodiments, the at leastone chemically modified nucleobase is selected from the group consistingof pseudouracil (ψ), N1-methylpseudouracil (m1ψ), 1-ethylpseudouracil,2-thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil,5-methoxyuracil, and any combination thereof. In some embodiments, atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 99%, or 100% of theuracils are N1-methylpseudouracils.

In some embodiments of the above aspect, the first mRNA comprises thenucleic acid sequence set forth in SEQ ID NO:5, and the second mRNAcomprises the nucleic acid sequence set forth in SEQ ID NO:6. In someembodiments, the first mRNA comprises the nucleic acid sequence setforth in SEQ ID NO:5, a 5′ terminal cap comprising Cap1, and a poly-Aregion 100 nucleotides in length, and the second mRNA comprises thenucleic acid sequence set forth in SEQ ID NO:6, a 5′ terminal capcomprising Cap1, and a poly-A region 100 nucleotides in length. In someembodiments, all of the uracils of the first polynucleotide and thesecond polynucleotide are N1-methylpseudouracils.

In some embodiments of the above aspect, the delivery agent comprises alipid nanoparticle comprising:

-   -   (i) Compound II, (ii) Cholesterol, and (iii) PEG-DMG or Compound        I;    -   (i) Compound VI, (ii) Cholesterol, and (iii) PEG-DMG or Compound        I;    -   (i) Compound II, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv)        PEG-DMG or Compound I;    -   (i) Compound VI, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv)        PEG-DMG or Compound I;    -   (i) Compound II, (ii) Cholesterol, and (iii) Compound I;    -   (i) Compound II, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv)        Compound I; or    -   (i) Compound II, (ii) DSPC, (iii) Cholesterol, and (iv) Compound        I.

In another aspect, the disclosure features a pharmaceutical compositioncomprising a first mRNA comprising a first open reading frame (ORF)encoding a first polypeptide comprising a heavy chain variable region ofan anti-chikungunya virus antibody and a second mRNA comprising a secondORF encoding a second polypeptide comprising a light chain variableregion of the anti-chikungunya virus antibody, wherein the firstpolypeptide and the second polypeptide pair to form the anti-chikungunyavirus antibody, and wherein the pharmaceutical composition whenadministered to a human subject in need thereof as a single doseadministration is sufficient to:

-   -   (i) protect the human subject from chikungunya virus infection,        after exposure to a chikungunya virus, for at least 24 hours, 48        hours, 72 hours, 96 hours, 168 hours, 336 hours, or 720 hours        after the single dose administration;    -   (ii) protect the human subject from onset of chikungunya fever,        after exposure to a chikungunya virus, for at least 24 hours, 48        hours, 72 hours, 96 hours, 168 hours, 336 hours, or 720 hours        after the single dose administration; and/or    -   (iii) provide systemic production of the anti-chikungunya virus        antibody in the human subject at a level of at least 5 μg/ml, 10        μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, or 30 μg/ml for at least 24        hours, 48 hours, 72 hours, 96 hours, 168 hours, 336 hours, or        720 hours after the single dose administration.

In some embodiments of the above aspect, the single dose administrationis an intravenous administration. In some embodiments of the aboveaspect, the single dose administration is a subcutaneous administration.

In some embodiments of the above aspect, the pharmaceutical compositionfurther comprises a delivery agent. In some embodiments, the deliveryagent comprises a lipid nanoparticle comprising:

-   -   (i) Compound II, (ii) Cholesterol, and (iii) PEG-DMG or Compound        I;    -   (i) Compound VI, (ii) Cholesterol, and (iii) PEG-DMG or Compound        I;    -   (i) Compound II, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv)        PEG-DMG or Compound I;    -   (i) Compound VI, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv)        PEG-DMG or Compound I;    -   (i) Compound II, (ii) Cholesterol, and (iii) Compound I;    -   (i) Compound II, (ii) DSPC or DOPE, (iii) Cholesterol, and (iv)        Compound I; or    -   (i) Compound II, (ii) DSPC, (iii) Cholesterol, and (iv) Compound        I.

In some embodiments of the above aspect, the first polypeptide comprisesthe heavy chain variable region of the heavy chain antibody sequence ofSEQ ID NO:1, and the second polypeptide comprises the light chainvariable region of the light chain antibody sequence of SEQ ID NO:3. Insome embodiments of the above aspect, the first polypeptide comprisesthe heavy chain constant region of the heavy chain antibody sequence ofSEQ ID NO:1, and the second polypeptide comprises the light chainconstant region of the light chain antibody sequence of SEQ ID NO:3.

In some embodiments of the above aspect, the first mRNA and the secondmRNA each comprise a microRNA (miR) binding site. In some embodiments,the microRNA is expressed in an immune cell of hematopoietic lineage ora cell that expresses TLR7 and/or TLR8 and secretes pro-inflammatorycytokines and/or chemokines. In some embodiments, the microRNA bindingsite is for a microRNA selected from the group consisting of miR-126,miR-142, miR-144, miR-146, miR-150, miR-155, miR-16, miR-21, miR-223,miR-24, miR-27, miR-26a, or any combination thereof. In someembodiments, the microRNA binding site is for a microRNA selected fromthe group consisting of miR126-3p, miR-142-3p, miR-142-5p, miR-155, orany combination thereof. In some embodiments, the microRNA binding siteis a miR-142-3p binding site. In some embodiments, the microRNA bindingsite is located in the 3′ UTR of the mRNA.

In some embodiments of the above aspect, the first mRNA and the secondmRNA each comprise a 5′ terminal cap. In some embodiments, each 5′terminal cap comprises a Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, 2-azidoguanosine, Cap2, Cap4, 5′methylG cap, or an analog thereof.

In some embodiments of the above aspect, the first mRNA and the secondmRNA each comprise a poly-A region. In some embodiments, each poly-Aregion is at least about 10, at least about 20, at least about 30, atleast about 40, at least about 50, at least about 60, at least about 70,at least about 80, at least about 90 nucleotides in length, or at leastabout 100 nucleotides in length. In some embodiments, each poly-A regionis about 10 to about 200, about 20 to about 180, about 50 to about 160,about 70 to about 140, or about 80 to about 120 nucleotides in length.

In some embodiments of the above aspect, the first mRNA and the secondmRNA each comprise at least one chemically modified nucleobase, sugar,backbone, or any combination thereof. In some embodiments, the at leastone chemically modified nucleobase is selected from the group consistingof pseudouracil (ψ), N-methylpseudouracil (m1ψ), 1-ethylpseudouracil,2-thiouracil (s2U), 4′-thiouracil, 5-methylcytosine, 5-methyluracil,5-methoxyuracil, and any combination thereof. In some embodiments, atleast about 25%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 99%, or 100% of theuracils are N1-methylpseudouracils.

In some embodiments of the above aspect, the human subject has achikungunya virus infection.

In another aspect, the disclosure features a method of treating achikungunya virus infection in a human subject that has been infectedwith a chikungunya virus, comprising administering to the human subjectan effective amount of a pharmaceutical composition disclosed herein ora polynucleotide disclosed herein.

In another aspect, the disclosure features a method of reducing thelikelihood of contracting a chikungunya virus infection in a humansubject in need thereof, comprising administering to the human subjectan effective amount of a pharmaceutical composition disclosed herein ora polynucleotide disclosed herein.

In another aspect, the disclosure features a method of preventing achikungunya virus infection in a human subject in need thereof,comprising administering to the human subject an effective amount of apharmaceutical composition disclosed herein or a polynucleotidedisclosed herein.

In another aspect, the disclosure features a method of expressing ananti-chikungunya virus antibody in a human subject in need thereof,comprising administering to the human subject an effective amount of apharmaceutical composition disclosed herein or a polynucleotidedisclosed herein.

In another aspect, the disclosure features a method of reducingchikungunya virus levels in a human subject in need thereof, comprisingadministering to the human subject an effective amount of apharmaceutical composition disclosed herein or a polynucleotidedisclosed herein.

In some embodiments of the above aspects, (i) the human subject isprotected from chikungunya virus infection, after exposure to thechikungunya virus, for at least 24 hours, 48 hours, 72 hours, 96 hours,168 hours, 336 hours, or 720 hours after a single dose administration;(ii) the human subject is protected from onset of chikungunya fever,after exposure to the chikungunya virus, for at least 24 hours, 48hours, 72 hours, 96 hours, 168 hours, 336 hours, or 720 hours after asingle dose administration; and/or (iii) systemic production of theanti-chikungunya virus antibody in the human subject is at a level of atleast 5 μg/ml, 10 μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, or 30 μg/ml forat least 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 336 hours,or 720 hours after a single dose administration.

In some embodiments of the above aspects, the pharmaceutical compositionor polynucleotide is administered to the human subject multiple times ata frequency of about once a week, about once every two weeks, or aboutonce a month. In some embodiments of the above aspects, thepharmaceutical composition or polynucleotide is administeredintravenously. In some embodiments of the above aspects, thepharmaceutical composition or polynucleotide is administeredsubcutaneously.

In another aspect, the disclosure features a pharmaceutical compositioncomprising: (i) a first polynucleotide comprising a first nucleic acidsequence encoding a first polypeptide comprising a heavy chain variableregion comprising the ChikV24 heavy chain CDR1, CDR2, and CDR3 sequences(amino acids 46-53 of SEQ ID NO:1, amino acids 71-78 of SEQ ID NO:1, andamino acids 117-131 of SEQ ID NO:1, respectively); and (ii) a secondpolynucleotide comprising a second nucleic acid sequence encoding asecond polypeptide comprising a light chain variable region comprisingthe ChikV24 light chain CDR1, CDR2, and CDR3 sequences (amino acids47-53 of SEQ ID NO:3, amino acids 71-73 of SEQ ID NO:3, and amino acids110-118 of SEQ ID NO:3, respectively), wherein the first polypeptidewhen paired with the second polypeptide forms an anti-chikungunya virusantibody or an anti-chikungunya virus antibody fragment. The antibody orantibody fragment may comprise the ChikV24 light and heavy chainvariable sequences (amino acids 21-128 of SEQ ID NO:3 and amino acids21-142 of SEQ ID NO:1, respectively). The antibody may be an IgG. Thepharmaceutical composition may comprise a delivery vehicle. The firstpolynucleotide and the second polynucleotide may each be DNA sequences,or may each be mRNA sequences. The first polynucleotide and the secondpolynucleotide may each comprise non-natural, modified nucleotides. Thefirst polynucleotide (e.g., an mRNA) and the second polynucleotide(e.g., an mRNA) may each comprise a heterologous 5′ UTR sequence. Thefirst polynucleotide (e.g., an mRNA) and the second polynucleotide(e.g., an mRNA) may each comprise a heterologous 3′ UTR sequence. Thefirst polynucleotide (e.g., an mRNA) and the second polynucleotide(e.g., an mRNA) may each comprise a heterologous 5′ UTR sequence and aheterologous 3′ UTR sequence. A “heterologous” UTR sequence is a UTRsequence other than a naturally occurring UTR sequence present in anaturally occurring mRNA that encodes an antibody heavy or light chaincomprising a ChikV24 variable region. Also provided is a method oftreating a human subject infected with chikungunya virus, or reducingthe likelihood of infection of a subject at risk of contractingchikungunya virus, comprising administering to the human subject aneffective amount of the pharmaceutical composition of this paragraph.

Each of the limitations of the disclosure can encompass variousembodiments of the disclosure. It is, therefore, anticipated that eachof the limitations of the disclosure involving any one element orcombinations of elements can be included in each aspect of thedisclosure. This disclosure is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The disclosureis capable of other embodiments and of being practiced or of beingcarried out in various ways.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1A is a graph showing the serum concentration levels of humanChikV24 antibody in AG129 mice 24 hours after intravenous administrationof 10 mg/kg, 2 mg/kg, or 0.4 mg/kg of the recombinant ChikV24 antibody.

FIG. 1B is a Kaplan-Meier survival plot showing the percent survival ofAG129 mice intravenously administered 10 mg/kg, 2 mg/kg, or 0.4 mg/kg ofpurified ChikV24 antibody or a control influenza antibody over thecourse of 21 days following challenge with virus. Survival data wereanalyzed using the Wilcoxon log-rank survival analysis. The number ofanimals in each group was 10.

FIG. 2A is a graph showing the serum concentration levels of humanChikV24 antibody in AG129 mice 24 hours after intravenous administrationof 0.5 mg/kg, 0.1 mg/kg, or 0.02 mg/kg of mRNAs encoding the heavy andlight chains of the ChikV24 antibody. The graph also shows the serumconcentration levels of a control influenza antibody 24 hours afterintravenous injection of 0.5 mg/kg of mRNAs encoding the controlantibody.

FIG. 2B is a graph showing the percent survival of AG129 miceintravenously administered 0.5 mg/kg (top line), 0.1 mg/kg (middleline), or 0.02 mg/kg (bottom line) of mRNAs encoding the heavy and lightchains of the ChikV24 antibody, or 0.5 mg/kg of mRNAs expressing acontrol antibody (dashed line), over the course of 21 days followingchallenge with virus. **(P<0.01) Indicates the survival differedsignificantly from that of the group treated with 0.5 mg/kg of theirrelevant control IgG (Wilcoxon log-rank survival test).

FIG. 2C is a graph showing the chikungunya virus titer in blood samplescollected from AG129 mice injected intravenously with 0.5 mg/kg, 0.1mg/kg, or 0.02 mg/kg of mRNAs expressing the heavy and light chains ofthe ChikV24 antibody, or 0.5 mg/kg of mRNAs expressing a controlantibody, two days after being challenged with virus. The mean valuesare indicated, and error bars show the standard deviation. Comparisonswere made by the Kruskal Wallis test with Dunn's post-test. ***indicates P<0.0003, as compared to mice injected with the control IgG.

FIG. 3 is a graph showing the serum concentrations of the ChikV24antibody from AG129 mice injected intravenously with 0.5 mg/kg, 0.1mg/kg, or 0.02 mg/kg of mRNAs expressing the heavy and light chains ofChikV24 antibody at 24-hours, 48-hours, or 72-hours post-injection.

FIG. 4A is a graph showing foot swelling as monitored by digitalcalipers in either C57BL/6 mice that were injected with 10 mg/kg ofmRNAs encoding human ChikV24 antibody, or control C57BL/6 mice that wereinjected with mRNAs encoding an antibody that does not bind tochikungunya virus, at 4 hours following inoculation with chikungunyavirus. The line indicates significance between the groups at each timepoint. Error bars indicate standard error of the mean.

FIG. 4B is a graph showing chikungunya virus RNA levels quantified byqRT-PCR in serum collected at 2 dpi from C57BL/6 mice that were injectedwith 10 mg/kg of mRNAs encoding human ChikV24 antibody (right area ofgraph), or control C57BL/6 mice that were injected with mRNAs encodingan antibody that does not bind to chikungunya virus (left area ofgraph), at 4 hours following inoculation with chikungunya virus. Barsindicate median values. Dotted lines indicate the limit of detection.

FIG. 4C is a graph showing chikungunya virus RNA levels quantified byqRT-PCR in ipsilateral (i.) and contralateral (c.) ankles that werecollected at 7 dpi from C57BL/6 mice that were injected with 10 mg/kg ofmRNAs encoding human ChikV24 antibody (right area of graph), or controlC57BL/6 mice that were injected with mRNAs encoding an antibody thatdoes not bind to chikungunya virus (left area of graph), at 4 hoursfollowing inoculation with chikungunya virus. Bars indicate medianvalues. Dotted lines indicate the limit of detection.

FIG. 4D contains histology section images taken from ipsilateral feetcollected at 7 dpi C57BL/6 mice that were injected with 10 mg/kg ofmRNAs encoding human ChikV24, or control C57BL/6 mice that were injectedwith mRNAs encoding an antibody that does not bind to chikungunya virus,at 4 hours following inoculation with chikungunya virus. Images showlow-magnification (scale bar 100 μm) with a high magnification inset(scale bar 10 μm). Top and bottom panels are representative images ofthe joint space and midfoot, respectively (n=5/group, two experiments).Arrows indicate cellular infiltrate in joint space.

FIG. 5A is a graph showing the serum concentration levels of the humanChikV24 antibody in cynomolgus monkeys injected intravenously with asingle 0.5 mg/kg dose of mRNAs expressing the heavy and light chains ofthe ChikV24 antibody over the course of 720-hours post-injection.

FIG. 5B is a graph showing ChikV24 antibody activity levels (μg/mL) inserum samples collected from cynomolgus monkeys 24 hours after infusionwith 0.5 mg/kg of mRNAs encoding the ChikV24 antibody, as measured usinga focus reduction neutralization assay (FRNT₅₀) and by ELISA.

FIG. 6 is a graph showing the serum concentration levels of the humanChikV24 antibody in cynomolgus monkeys injected intravenously with two0.3 mg/kg, 1 mg/kg, or 3 mg/kg doses of mRNAs expressing the heavy andlight chains of the ChikV24 antibody over the course of 2400-hours (100days) post-injection.

DETAILED DESCRIPTION

Described herein are compositions for the prevention or treatment ofdiseases or symptoms associated with chikungunya virus (CHIKV)infection, e.g., chikungunya fever. RNA therapeutics are well-suited forthe prevention or treatment of chikungunya fever, as the technologyprovides for the intracellular delivery of mRNAs encoding the heavy andlight chain polypeptides of an anti-CHIKV antibody, followed by de novosynthesis of functional anti-CHIKV antibody within target cells. Afterdelivery of mRNA to the target cells, the anti-CHIKV antibody proteinsare expressed by the cells' own translational machinery, and hence,fully functional antibody can bind to and neutralize the chikungunyavirus, thereby preventing further viral infection.

As described herein, the disclosure provides a ribonucleic acid (RNA)polynucleotide having an open reading frame encoding a heavy chainpolypeptide of an antibody, or a portion thereof (e.g., a heavy chainpolypeptide variable region), having specificity for a chikungunya virusprotein and a pharmaceutically acceptable carrier or excipient. In someembodiments, the disclosure provides an RNA polynucleotide having anopen reading frame encoding a light chain polypeptide of an antibody, ora portion thereof (e.g., a light chain polypeptide variable region),having specificity for a chikungunya virus protein and apharmaceutically acceptable carrier or excipient.

Described herein are compositions (including pharmaceuticalcompositions) and methods for the design, preparation, manufactureand/or formulation of antibodies with specificity for CHIKV, wherein atleast one component of the antibody is encoded by a polynucleotide. Assuch the present invention is directed, in part, to polynucleotides,specifically IVT polynucleotides, chimeric polynucleotides and/orcircular polynucleotides encoding one or more anti-CHIKV antibodiesand/or components thereof.

The methods of the present invention are and can be used to engineernovel polynucleotides for the in vivo production of antibodies in such amanner as to provide improvements over standard antibody technology. Insome cases, the polynucleotides provided herein encode antibodies, orportions thereof, that have been designed to produce a therapeuticoutcome and optionally improve one or more of the stability and/orclearance in tissues, receptor uptake and/or kinetics, cellular access,engagement with translational machinery, mRNA half-life, translationefficiency, protein production capacity, secretion efficiency (whenapplicable), accessibility to circulation, protein half-life and/ormodulation of a cell's status, antibody target affinity and/orspecificity, reduction of antibody cross reactivity, increase ofantibody purity, increase or alteration of antibody effector functionand/or antibody activity.

1. Antibodies Specific for Chikungunya Virus

The polynucleotides, constructs, and/or compositions of the presentdisclosure are useful for producing antibodies that bind to achikungunya virus (CHIKV), e.g., to a CHIKV antigenic polypeptide.

In some embodiments the compositions and methods are useful for theprevention, treatment, or management of CHIKV infection, e.g.,chikungunya fever. Some embodiments of the present disclosure provideRNA polynucleotides, e.g., mRNA, encoding an anti-CHIKV antibody,fragment, or variant thereof, which may be used to treat or preventchikungunya fever. In some embodiments, one or more RNA polynucleotideshave open reading frames (ORFs) encoding at least one anti-CHIKVantibody that binds specifically to a CHIKV antigenic polypeptide. Insome embodiments, the RNA polynucleotides encode two or more anti-CHIKVantibodies. In some embodiments, two or more RNA polynucleotides, e.g.,two or more mRNAs, encode portions or fragments of an anti-CHIKVantibody. For example, one mRNA polynucleotide can have an ORF encodinga heavy chain of the anti-CHIKV antibody, and one mRNA polynucleotidecan have an ORF encoding a light chain of the anti-CHIKV antibody, suchthat the two mRNAs in combination express the heavy and light chainpolypeptides that together form the antibody, e.g., in a cell. In someembodiments, the mRNA polynucleotides described herein encode anantibody that neutralizes CHIKV.

An antibody is an immunoglobulin molecule capable of specific binding toa target through at least one antigen recognition site, located in thevariable region of the immunoglobulin molecule. Most antibodies comprisetwo heavy chains and two light chains. There are several different typesof antibody heavy chains, and several different kinds of antibodies,which are grouped into different isotypes based on which heavy chainthey possess. Five different antibody isotypes (IgA, IgD, IgE, IgG andIgM) are known in mammals and trigger a different immune response foreach different type of foreign object, epitope or microbe theyencounter. The antibodies described herein can be derived from murine,rat, human, or any other origin. The majority of antibodies aregenerated using recombinant or cloning strategies and productheterogeneity is common to monoclonal antibody and other recombinantbiological production. Such heterogeneity is typically introduced eitherupstream during expression or downstream during manufacturing.Recombinant antibody engineering involves the use of viruses or yeast tocreate antibodies, rather than mice which are used in cloningstrategies. All of these however, suffer from drawbacks associated withthe systems used for generation including degree of purity, speed ofdevelopment, cross reactivity, low affinity and variable specificity.

As used herein, the term “antibody” encompasses not only intact (i.e.,full-length) antibodies, but also antigen-binding fragments thereof(such as Fab, Fab′, F(ab′)2, Fv), single chain (scFv), mutants thereof,fusion proteins comprising an antibody portion, humanized antibodies,chimeric antibodies, diabodies, nanobodies, linear antibodies, singlechain antibodies, multispecific antibodies (e.g., bispecificantibodies), single domain antibodies such as heavy-chain antibodies,and any other modified configuration of the immunoglobulin molecule thatcomprises an antigen recognition site of the required specificity,including glycosylation variants of antibodies, amino acid sequencevariants of antibodies, and covalently modified antibodies. An antibodyincludes an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM(or sub-class thereof), and the antibody need not be of any particularclass. Depending on the antibody's amino acid sequence of the constantdomain of its heavy chains (if applicable), immunoglobulins can beassigned to different classes. There are five major classes ofnaturally-occurring immunoglobulins: IgA, IgD, IgE, IgG, and IgM, andseveral of these may be further divided into subclasses (isotypes),e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy-chain constantdomains that correspond to the different classes of immunoglobulins arecalled alpha, delta, epsilon, gamma, and mu, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

An antibody described herein may comprise a heavy chain variable region(V_(H)), a light chain variable region (V_(L)), or a combinationthereof. Optionally, the antibody may further comprise an antibodyconstant region or a portion thereof (e.g., C_(H)1, C_(H)2, C_(H)3, or acombination thereof). The heavy chain constant region can be of anysuitable class as described herein and of any suitable origin, e.g.,human, mouse, rat, or rabbit. In one specific example, the heavy chainconstant region is derived from a human IgG (a gamma heavy chain). Thelight chain constant region can be a kappa chain or a lambda chain froma suitable origin. Antibody heavy and light chain constant regions arewell known in the art, e.g., those provided in the IMGT database(www.imgt.org) or at www.vbase2.org/vbstat.php., both of which areincorporated by reference herein.

In some embodiments, the antibodies described herein specifically bindto the corresponding target antigen or an epitope thereof. An antibodythat “specifically binds” to an antigen or an epitope is a term wellunderstood in the art. A molecule is said to exhibit “specific binding”if it reacts more frequently, more rapidly, with greater duration and/orwith greater affinity with a particular target antigen than it does withalternative targets. An antibody “specifically binds” to a targetantigen or epitope if it binds with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other substances.For example, an antibody that specifically (or preferentially) binds toan antigen (e.g., a viral antigen) or an antigenic epitope therein is anantibody that binds this target antigen with greater affinity, avidity,more readily, and/or with greater duration than it binds to otherantigens or other epitopes in the same antigen. It is also understoodwith this definition that, for example, an antibody that specificallybinds to a first target antigen may or may not specifically orpreferentially bind to a second target antigen. As such, “specificbinding” or “preferential binding” does not necessarily require(although it can include) exclusive binding. In some examples, anantibody that “specifically binds” to a target antigen or an epitopethereof may not bind to other antigens or other epitopes in the sameantigen.

In some embodiments, the mRNA polynucleotides described herein encode anantibody that binds to CHIKV. The mRNAs of the present disclosure canencode one or more polypeptides that form an antibody, or anantigen-binding portion thereof, that specifically binds to andneutralizes CHIKV. In one exemplary embodiment, mRNA polynucleotidesdescribed herein encode a heavy chain polypeptide of an antibody, alight chain polypeptide of an antibody, or heavy and light chainpolypeptides of an antibody. In exemplary aspects, polynucleotides ofthe disclosure, e.g., polynucleotides encoding an anti-CHIKV antibody orportion thereof, may include at least one chemical modification.

Chikungunya virus is a positive-sense single-stranded RNA alphavirusthat is approximately 60-70 nm in diameter. The virion consists of anenvelope and a nucleocapsid. The chikungunya virus genome isapproximately 11.7 to 11.8 kb and encodes four nonstructural proteins(the nsP1, nsP2, nsP3 and nsP4 proteins), and five structural proteins(the capsid (C) protein, three envelope proteins (E1), (E2), and (E3),and the 6K protein). The structural proteins are translated from asubgenomic 26S mRNA as a single polyprotein. This polyprotein isprocessed into the five structural proteins. The four nonstructuralproteins are also processed from a single polyprotein. Severalchikungunya virus strains have been isolated and sequenced, and can befound at, e.g., NCBI GenBank Accession Nos: NC_004162.2, MF580946,AF369024, EU037962, KX702402, JF274082.1, KY038947.2, KY038946.1, andDQ443544.1.

In some embodiments, the anti-CHIKV antibodies described herein can bindto an antigenic polypeptide of CHIKV. In some embodiments, theanti-CHIKV antibodies described herein can bind to an antigenicpolypeptide of any CHIKV strain. In some embodiments, the anti-CHIKVantibody binds specifically to an antigenic polypeptide which is a CHIKVstructural protein or an antigenic fragment thereof. For example, aCHIKV structural protein may be an envelope protein (E), a 6K protein,or a capsid (C) protein. In some embodiments, the CHIKV structuralprotein is an envelope protein selected from E1, E2, and E3. In someembodiments, the CHIKV structural protein is E1 or E2. In someembodiments, the CHIKV structural protein is a capsid protein. In someembodiments, the antigenic polypeptide is a fragment or epitope of aCHIKV structural protein.

In some embodiments, an antibody described herein binds to an epitope onsurface of the CHIKV capsid and/or envelope. In some embodiments, anantibody described herein binds to an epitope on the E2 protein ofCHIKV. In some embodiments, the antibody binds to E2-A162, or an epitopeformed by residues E2-G95, E2-A162, E2-A164, E2-E165, E2-E166 and/orE2-I167, or any combination thereof. In some embodiments, the antibodybinds to an epitope formed by residues E2-Y69, E2-F84, E2-V113, E2-G114,E2-T116, and/or E2-D117, or any combination thereof. In someembodiments, the epitope comprises E2-G95.

In some embodiments, an antibody described herein binds to at least oneof: Subunit I-E2-E24 and Subunit I-E2-I121 and at least one of: SubunitII-E2-G55, Subunit II-E2-W64, Subunit II-E2-K66, Subunit II-E2-R80. Insome embodiments, the antibody binds to Subunit I-E2-E24 and SubunitI-E2-I121 and at least one of: Subunit II-E2-G55, Subunit II-E2-W64,Subunit II-E2-K66, Subunit II-E2-R80. In some embodiments, the antibodybinds to at least two of Subunit II-E2-G55, Subunit II-E2-W64, SubunitII-E2-K66, Subunit II-E2-R80. In some embodiments, the antibody binds toat least three of Subunit II-E2-G55, Subunit II-E2-W64, SubunitII-E2-K66, Subunit II-E2-R80. In some embodiments, the antibody binds toat least three of Subunit II-E2-G55, Subunit II-E2-W64, SubunitII-E2-K66, and Subunit II-E2-R80. In some embodiments, the antibodybinds to Subunit II-E2-G55, Subunit II-E2-W64, Subunit II-E2-K66, andSubunit II-E2-R80.

In some embodiments, the antibody binds to the membrane distal region ofa CHIKV E1/E2 trimer. In some embodiments, the antibody binds to theexterior face of the E1/E2 heterocomplex. The exterior face refers tothe portion of the E1/E2 heterocomplex that is exposed when the E1/E2hetero-protein is in its native form on the virion surface, such as inits trimeric form.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a heavy chain polypeptide having an amino acid sequencesharing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity with SEQ IDNO: 1. In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a heavy chain polypeptide having an amino acid sequencethat is identical to SEQ ID NO: 1. In some embodiments, the antibodiesor antigen binding fragments thereof, have a heavy chain polypeptidehaving an amino acid sequence differing by up to 20 amino acids from SEQID NO: 1, e.g., differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acids from SEQ ID NO: 1.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a heavy chain variable region having an amino acidsequence sharing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity withamino acids 21-142 of SEQ ID NO: 1. In some embodiments, the antibodies,or antigen binding fragments thereof, have a heavy chain variable regionhaving an amino acid sequence that is identical to amino acids 21-142 ofSEQ ID NO: 1.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a heavy chain constant region having an amino acidsequence sharing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity withamino acids 143-472 of SEQ ID NO: 1. In some embodiments, theantibodies, or antigen binding fragments thereof, have a heavy chainconstant region having an amino acid sequence that is identical to aminoacids 143-472 of SEQ ID NO: 1.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a signal sequence having an amino acid sequence sharing atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97% 98%, or 99% identity with amino acids 1-20 ofSEQ ID NO: 1. In some embodiments, the antibodies, or antigen bindingfragments thereof, have a signal sequence having an amino acid sequencethat is identical to amino acids 1-20 of SEQ ID NO: 1.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a light chain polypeptide having an amino acid sequencesharing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity with SEQ ID NO:3. In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a light chain polypeptide having an amino acid sequencethat is identical to SEQ ID NO: 3. In some embodiments, the antibodiesor antigen binding fragments thereof, have a light chain polypeptidehaving an amino acid sequence differing by up to 20 amino acids from SEQID NO: 3, e.g., differing by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acids from SEQ ID NO: 3.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a light chain variable region having an amino acidsequence sharing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity withamino acids 21-128 of SEQ ID NO: 3. In some embodiments, the antibodies,or antigen binding fragments thereof, have a light chain variable regionhaving an amino acid sequence that is identical to amino acids 21-128 ofSEQ ID NO: 3.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a light chain constant region having an amino acidsequence sharing at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, or 99% identity withamino acids 129-235 of SEQ ID NO: 3. In some embodiments, theantibodies, or antigen binding fragments thereof, have a light chainconstant region having an amino acid sequence that is identical to aminoacids 129-235 of SEQ ID NO: 3.

In some embodiments, the antibodies, or antigen binding fragmentsthereof, have a signal sequence having an amino acid sequence sharing atleast 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97% 98%, or 99% identity with amino acids 1-20 ofSEQ ID NO: 3. In some embodiments, the antibodies, or antigen bindingfragments thereof, have a signal sequence having an amino acid sequencethat is identical to amino acids 1-20 of SEQ ID NO: 3.

The mRNA polynucleotides described herein may be designed to encodeknown antibodies, or antigen-binding fragments, such as Fab fragments,that bind to CHIKV, e.g., as described in Porta et al., 2016, J. Virol.90(3): 1169-1177. In other embodiments, the mRNA polynucleotides encodevariants of known antibodies, or antigen-binding fragments, such as Fabfragments, that bind to CHIKV.

In some examples, the antibody, or antigen binding portion thereof,binds the same chikungunya virus epitope as an antibody, orantigen-binding portion thereof, known in the art and/or exemplifiedherein and/or competes against such an antibody from binding to theantigen. Such an antibody may comprise the same heavy chain CDRs asthose known in the art and/or exemplified herein. An antibody having thesame CDR (e.g., CDR3) as a reference antibody, or antigen-bindingportion thereof, means that the two antibodies have the same amino acidsequence in that CDR region as determined by the same methodology (e.g.,the Kabat definition, the Chothia definition, the AbM definition, or thecontact definition).

Alternatively, an antibody, or antigen-binding portion thereof,described herein may comprise up to 5 (e.g., 4, 3, 2, or 1) amino acidresidue variations in one or more of the CDR regions of one of theantibodies, or antigen-binding portions thereof, known in the art and/orexemplified herein and binds the same epitope of antigen withsubstantially similar affinity (e.g., having a KD value in the sameorder). In one example, the amino acid residue variations areconservative amino acid residue substitutions. As used herein, a“conservative amino acid substitution” refers to an amino acidsubstitution that does not alter the relative charge or sizecharacteristics of the protein in which the amino acid substitution ismade. Variants can be prepared according to methods for alteringpolypeptide sequence known to one of ordinary skill in the art such asare found in references which compile such methods, e.g., MolecularCloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989,or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

In some embodiments, the mRNA polynucleotides described herein encodeone or more antibodies, or combinations of antibodies, selected from thegroup consisting of IgA, IgG, IgM, IgE, and IgD, that can bindspecifically to CHIKV.

In some embodiments, a variable domain of the antibodies describedherein comprises three complementarity determining regions (CDRs), eachof which is flanked by a framework region (FW). For example, a VH domainmay comprise a set of three heavy chain CDRs, HCDR1, HCDR2, and HCDR3. AVL domain may comprise a set of three light chain CDRs, LCDR1, LCDR2,and LCDR3. A set of HCDRs can be provided in a VH domain that is used incombination with a VL domain. A VH domain may be provided with a set ofHCDRs, and if such a VH domain is paired with a VL domain, then the VLdomain may be provided with a set of LCDRs disclosed herein.

In some embodiments, an antibody as described herein has a suitablebinding affinity for the target antigen or antigenic epitopes thereof,e.g., an antigenic polypeptide or epitope of CHIKV. As used herein,“binding affinity” refers to the apparent association constant or KA.The KA is the reciprocal of the dissociation constant (KD). The antibodydescribed herein may have a binding affinity (KD) of at least 10−5,10−6, 10−7, 10−8, 10−9, 10−10 M, or lower for the target antigen orantigenic epitope. An increased binding affinity corresponds to adecreased KD. Higher affinity binding of an antibody for a first antigenrelative to a second antigen can be indicated by a higher KA (or asmaller numerical value KD) for binding the first antigen than the KA(or numerical value KD) for binding the second antigen. In such cases,the antibody has specificity for the first antigen (e.g., a firstprotein in a first conformation or mimic thereof) relative to the secondantigen (e.g., the same first protein in a second conformation or mimicthereof; or a second protein).

For example, in some embodiments, the chikungunya virus antibodiesdescribed herein have a higher binding affinity (a higher KA or smallerKD) to a first chikungunya virus strain or as compared to the bindingaffinity to a second chikungunya virus strain. Differences in bindingaffinity (e.g., for specificity or other comparisons) can be at least1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000,10,000 or 105 fold. In some embodiments, any of the anti-chikungunyavirus antibodies may be further affinity matured to increase the bindingaffinity of the antibody to the target antigen or antigenic epitopethereof.

Binding affinity (or binding specificity) can be determined by a varietyof methods including equilibrium dialysis, equilibrium binding, gelfiltration, ELISA, surface plasmon resonance, or spectroscopy (e.g.,using a fluorescence assay). Exemplary conditions for evaluating bindingaffinity are in HBS-P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005%(v/v) Surfactant P20). These techniques can be used to measure theconcentration of bound binding protein as a function of target proteinconcentration. The concentration of bound binding protein ([Bound]) isgenerally related to the concentration of free target protein ([Free])by the following equation:[Bound]=[Free]/(Kd+[Free])

It is not always necessary to make an exact determination of KA or KDthough, since sometimes it is sufficient to obtain a quantitativemeasurement of affinity, e.g., determined using a method such as ELISAor FACS analysis, is proportional to KA or KD, and thus can be used forcomparisons, such as determining whether a higher affinity is, e.g.,2-fold higher, to obtain a qualitative measurement of affinity, or toobtain an inference of affinity, e.g., by activity in a functionalassay, e.g., an in vitro or in vivo assay.

In some embodiments, the antibody described herein is a humanizedantibody. Humanized antibodies refer to forms of non-human (e.g.,murine) antibodies that are specific chimeric immunoglobulins,immunoglobulin chains, or antigen-binding fragments thereof that containminimal sequence derived from non-human immunoglobulin. For the mostpart, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a complementary determining region(CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat, or rabbit havingthe desired specificity and/or affinity. In some instances, one or moreFv framework region (FR) residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, the humanizedantibody may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences, but areincluded to further refine and optimize antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the CDR regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the FR regions are thoseof a human immunoglobulin sequence (e.g., a germline sequence or aconsensus sequence). The humanized antibody optimally may also compriseat least a portion of an immunoglobulin constant region or domain (Fc),typically that of a human immunoglobulin. Antibodies may have Fc regionsmodified as described in WO 99/58572. Other forms of humanizedantibodies have one or more CDRs (one, two, three, four, five, and/orsix) which are altered with respect to the original antibody (termed oneor more CDRs “derived from” one or more CDRs from the originalantibody). Humanized antibodies may also involve optimized antibodiesderived from affinity maturation.

In another example, the antibody as described herein is a chimericantibody, which can include a heavy constant region and optionally alight constant region from a human antibody. Chimeric antibodies referto antibodies having a variable region or part of variable region from afirst species and a constant region from a second species. Typically, inthese chimeric antibodies, the variable region of both light and heavychains mimics the variable regions of antibodies derived from onespecies of mammals (e.g., a non-human mammal such as mouse, rabbit, andrat), while the constant portions are homologous to the sequences inantibodies derived from another mammal such as human. In someembodiments, amino acid modifications can be made in the variable regionand/or the constant region.

In yet another example, the antibody described herein can be asingle-domain antibody, which interacts with the target antigen via onlyone single variable domain such as a single heavy chain domain (asopposed to traditional antibodies, which interact with the targetantigen via heavy chain and light chain variable domains). A singledomain construct comprises one or two polynucleotides encoding a singlemonomeric variable antibody domain. In some cases, single domainantibodies comprise one variable domain (VH) of a heavy-chain antibody,and can be devoid of a light chain. In additional to a variable region(for example, a VH), a single-domain antibody may further comprise aconstant region, for example, C_(H)1, C_(H)2, C_(H)3, C_(H)4, or acombination thereof.

In some examples, the antibody is a single chain antibody, which maycomprise only one variable region (e.g., V_(H)) or comprise both a V_(H)and a V_(L). Such an antibody can be encoded by a single RNA molecule.In other examples, the antibody described herein is a multi-chainantibody comprising an independent heavy chain and an independent lightchain. Such a multi-chain antibody may be encoded by a singleribonucleic acid (RNA) molecule, which can be a bicistronic moleculeencoding two separate polypeptide chains. Such an RNA molecule maycontain a signal sequence between the two coding sequences such that twoseparate polypeptides would be produced in the translation process.Alternatively, the RNA molecule may include a sequence coding for acleavage site (e.g., a protease cleavage site) between the heavy andlight chains such that it produces a single precursor polypeptide, whichcan be processed via cleavage at the cleavage site to produce the twoseparate heavy and light chains. Alternatively, the heavy and lightantibody chains may be encoded by two separate RNA molecules, e.g., twoseparate mRNA molecules.

In some embodiments, the antibodies and antigen binding fragmentsthereof encoded by an RNA polynucleotide of the present applicationcomprises a fragment crystallizable (Fc) region. The Fc region is thetail region of an antibodies and antigen binding fragments thereof whichcontains constant domains (e.g., CH2 and CH3); the other region of theantibodies and antigen binding fragments thereof being the Fab regionwhich contains a variable domain (e.g., VH) and a constant domain (e.g.,CH1), the former of which defines binding specificity.

As described herein, antibodies can comprise a VH domain. In someembodiments, the VH domain further comprises one or more constantdomains (e.g., CH2 and/or CH3) of an Fc region and/or one or moreconstant domains (e.g., CH1) of a Fab region. In some embodiments, eachof the one or more constant domains (e.g., CH1, CH2, and/or CH3) cancomprise or consist of portions of a constant domain. For example, insome embodiments, the constant domain comprises 99% or less, 98% orless, 97% or less, 96% or less, 95% or less, 90% or less, 80% or less,70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% orless, or 10% or less of the corresponding full sequence.

In some embodiments the polynucleotides encode a single chain Fv (scFv)that binds to CHIKV. As used herein, the term “single-chain” refers to amolecule comprising amino acid monomers linearly linked by peptidebonds, e.g., a single chain Fv construct can be a polynucleotideencoding at least two coding regions and a linker region. The scFvconstruct may encode a fusion protein of the variable regions of theheavy (VH) and light chains (VL) of immunoglobulins, connected with ashort linker peptide of ten to about 25 amino acids. The linker can berich in glycine for flexibility, as well as serine or threonine forsolubility, and can either connect the N-terminus of the VH with theC-terminus of the VL, or vice versa. Other linkers include those knownin the art and disclosed herein. In some embodiments, an scFv has avariable domain of light chain (VL) connected from its C-terminus to theN-terminal end of a variable domain of heavy chain (VH) by a polypeptidechain. Alternately the scFv comprises of polypeptide chain where in theC-terminal end of the VH is connected to the N-terminal end of VL by apolypeptide chain. In some embodiments the scFv constructs may beoriented in a variety of ways. For instance, the order to VH and VL inthe construct may vary and alter the expression and/or activity of thescFv. In some embodiments the scFv constructs are oriented, from N to Cterminus, VL-linker-VH-linker-CH2-CH3.

In some embodiments, one or more flexible linkers can be used to linktwo or more portions or fragments of an antibody. For example, flexiblelinkers can attach scFv fragments to one another, and/or to Fc domains.In some embodiments, the variable heavy and variable light chains arecovalently attached using flexible linkers. In some embodiments,flexible linkers such as those containing glycine and serine are used.The scFV-FC synthesis from a typical antibody format can involve theaddition of linkers. In some embodiments, the linker is (GxS)3, whereinx can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some cases, longer linkersare more effective in producing highly neutralizing scFv, and increasingthe VL-VH linker length could reduce strain and oligomerization. In somecases, it is desirable to have a linker that is 15 amino acids orgreater in length, e.g., a linker that is 15 to 30 amino acids, 16-25amino acids, 20 or to 30 amino acids in length. The (G4S)4 linker (alsoreferred to as Linker20) is a longer linker that can less strain in somecases. In some embodiments the linker is (G4S)4

Examples of linkers which may be used in the polynucleotides of thepresent invention include those in Table 1.

TABLE 1 Linkers SEQ ID Name Sequence in polynucleotide NO PLrigidGAAGCTGCTGCAAGAGAAGCT 206 PLrigid is a 20 a.a. peptide thatGCAGCTAGGGAGGCTGCAGCT is based on an alpha-helix motifAGGGAGGCTGCTGCAAGA (EAAAR (SEQ ID NO: 205))(Merutka et al., 1991; Sommese et al., 2010) 2aa GS linker GGCAGC 207Highly flexibly glycine linker 6aa [GS]x linker (SEQ ID NO:GGTAGCGGCAGCGGTAGC 209 208) Highly flexible 6 amino acidlinker. Translates to gsgsgs (SEQ ID NO: 208). Codon-optimize forE. coli, yeast, mammalian 10 aa flexible protein domainGGTGAAAATTTGTATTTTCAAT 210 linker CTGGTGGT 8 aa protein domain linkerTCCGCTTGTTACTGTGAGCTTT 211 CC 15 aa flexible glycine-serineGGTGGAGGAGGTTCTGGAGGC 212 protein domain linker; FreiburgGGTGGAAGTGGTGGCGGAGGT standard AGC Short Linker (Gly-Gly-Ser-GGTGGTTCTGGT 214 Gly (SEQ ID NO: 213)) Middle Linker (Gly-Gly-Ser-GGTGGTTCTGGTGGTGGTTCTG 216 Gly)x2 (SEQ ID NO: 215) GTLong Linker (Gly-Gly-Ser- GGTGGTTCTGGTGGTGGTTCTG 218Gly)x3 (SEQ ID NO: 217) GTGGTGGTTCTGGT GSAT Linker GGTGGTTCTGCCGGTGGCTCC219 GGTTCTGGCTCCAGCGGTGGC AGCTCTGGTGCGTCCGGCACG GGTACTGCGGGTGGCACTGGCAGCGGTTCCGGTACTGGCTCT GGC SEG-Linker GGTGGTTCTGGCGGCGGTTCT 220GAAGGTGGCGGCTCCGAAGGC GGCGGCAGCGAGGGCGGTGGT AGCGAAGGTGGTGGCTCCGAGGGTGGCGGTTCCGGCGGCGGT AGC GGGGSGGGGSGGGGSGGGGS 221

Table references: Merutka G, Shalongo W, Stellwagen E. (1991) A modelpeptide with enhanced helicity. Biochem. 30: 4245-4248 and Sommese R F,Sivaramakrishnan S, Baldwin R L, Spudich J A. (2010) Helicity of shortE-R/K peptides. Protein Sci. 19: 2001-2005.

During construction of scFv initially, constant domains are removed. Twolinkers are added to connect the FV region which binds the antigen, andthe FC region. In some embodiments the FC region is a wild type of FCregion. In other embodiments it is a variant of wild type. In someembodiments a wild type constant region is a wild type IgG1 constantregion (e.g., ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:222)).

In some instances, the antibody may be a germlined variant of any of theexemplary antibodies disclosed herein. A germlined variant contains oneor more mutations in the framework regions as relative to its parentantibody towards the corresponding germline sequence. To make a germlinevariant, the heavy or light chain variable region sequence of the parentantibody or a portion thereof (e.g., a framework sequence) can be usedas a query against an antibody germline sequence database (e.g.,www.bioinfo.org.uk/abs/, www.vbase2.org, or www.imgt.org) to identifythe corresponding germline sequence used by the parent antibody andamino acid residue variations in one or more of the framework regionsbetween the germline sequence and the parent antibody. One or more aminoacid substitutions can then be introduced into the parent antibody basedon the germline sequence to produce a germlined variant.

The mRNA polynucleotides described herein can encode antibodies, orantigen-binding fragments thereof, with modified or variant variabledomains and/or constant domains compared to sequences disclosed herein.In some cases, modifications or variations can include amino acidsubstitutions, amino acid deletions, or amino acid additions, comparedto the sequences disclosed herein. The deleted amino acids typically maybe from the carboxyl or amino terminal ends of the heavy chain variableregion (VH) and/or the light chain variable region (VL).

When needed, the antibody as described herein may comprise a modifiedconstant region. For example, it may comprise a modified constant regionthat is immunologically inert, e.g., does not trigger complementmediated lysis, or does not stimulate antibody-dependent cell mediatedcytotoxicity (ADCC). ADCC activity can be assessed using methodsdisclosed in U.S. Pat. No. 5,500,362. Alternatively, the constant regionmay be modified such that it has an elevated effort activity, forexample, enhanced ADCC activity. In some embodiments, the constantregion can be modified as described in Eur. J. Immunol. (1999)29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK PatentApplication No. 9809951.8.

In some embodiments, the heavy chain constant region used in theantibodies described herein may comprise mutations (e.g., amino acidresidue substitutions) to enhance a desired characteristic of theantibody, for example, increasing the binding activity to the neonatalFc receptor (FcRn) and thus the serum half-life of the antibodies. Itwas known that binding to FcRn is critical for maintaining antibodyhomeostasis and regulating the serum half-life of antibodies. One ormore (e.g., 1, 2, 3, 4, 5, or more) mutations (e.g., amino acid residuesubstitutions) may be introduced into the constant region at suitablepositions (e.g., in C_(H)2 region) to enhance FcRn binding and enhancethe half-life of the antibody. See, e.g., Dall'Acqua et al., J.B.C.,2006, 281:23514-23524; Robbie et al., Antimicrob. Agents Chemother,2013, 57(12):6147; and Dall'Acqua et al., J. Immunol. 2002169:5171-5180.

In some embodiments, a polynucleotide is an intrabody construct whichhas been modified for expression inside a target cell and where theexpression product binds an intracellular protein. Such constructs mayhave sub picomolar binding affinities and may be formulated fortargeting to particular sites or tissues. For example, intrabodyconstructs may be formulated in any of the lipid nanoparticleformulations disclosed herein.

In some embodiment, the polynucleotide is a bicistronic constructencoding a two-protein chain antibody on a single polynucleotide strand.A pseudo-bicistronic construct is a polynucleotide encoding a singlechain antibody discontinuously on a single polynucleotide strand. Forbicistronic constructs, the encoded two strands or two portions/regionsand/or domains (as is the case with pseudo-bicistronic) are separated byat least one nucleotide not encoding the strands or domains. More oftenthe separation comprises a cleavage signal or site or a non-codingregion of nucleotides. Such cleavage sites include, for example, furincleavage sites encoded as an “RKR” site in the resultant polypeptide.

In some embodiments the antibodies are administered to a subject as abolus IV injection or bolus. This form of delivery can produce highlevels of expressed antibody.

2. Polynucleotides and Open Reading Frames

In some aspects, the polynucleotides disclosed herein are or function asa messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA)refers to any polynucleotide which encodes at least one peptide orpolypeptide of interest and which is capable of being translated toproduce the encoded peptide polypeptide of interest in vitro, in vivo,in situ or ex vivo. The basic components of an mRNA molecule typicallyinclude at least one coding region, a 5′ untranslated region (UTR), a 3′UTR, a 5′ cap, and a poly-A tail.

The instant invention features mRNAs for use in treating or preventingCHIKV infection in a subject. The mRNAs featured for use in theinvention are administered to subjects and encode a human anti-CHIKVantibody in vivo. Accordingly, the invention relates to polynucleotides,e.g., mRNA, comprising an open reading frame of linked nucleosidesencoding a human anti-CHIKV antibody polypeptide, functional fragmentsthereof, and fusion proteins. In some embodiments, the open readingframe is sequence-optimized. In particular embodiments, the inventionprovides sequence-optimized polynucleotides comprising nucleotidesencoding a polypeptide sequence of a human anti-CHIKV antibody, or aportion or fragment thereof, e.g., nucleotides encoding a heavy chain ora light chain of an anti-CHIKV antibody.

In certain aspects, the invention provides polynucleotides (e.g., a RNAsuch as an mRNA) that comprise a nucleotide sequence (e.g., an ORF)encoding one or more anti-CHIKV antibody polypeptides. In someembodiments, the encoded anti-CHIKV antibody polypeptide of theinvention can be selected from:

-   -   (i) a full length anti-CHIKV heavy chain polypeptide or a full        length anti-CHIKV light chain polypeptide;    -   (ii) a functional fragment of an anti-CHIKV heavy chain or light        chain polypeptide described herein (e.g., a truncated sequence        shorter than the heavy or light chain; but still retaining CHIKV        binding activity);    -   (iii) a variant (e.g., full length or truncated protein in which        one or more amino acids have been replaced) with respect to a        reference protein, e.g., a heavy chain (e.g., SEQ ID NO: 1) or        light chain (e.g., SEQ ID NO: 3) of an anti-CHIKV antibody; or    -   (iv) a fusion protein comprising (i) a full length heavy chain        (e.g., SEQ ID NO: 1), a functional fragment or a variant        thereof, or (ii) a full length light chain (e.g., SEQ ID NO: 3),        a functional fragment or a variant thereof; and (ii) a        heterologous protein.

In certain embodiments, the encoded polypeptide is a mammaliananti-CHIKV antibody polypeptide, such as a human anti-CHIKV antibodypolypeptide, a functional fragment or a variant thereof.

In some embodiments, one or more mRNA polynucleotide as described hereinexpresses an anti-CHIKV antibody in a mammalian cell, e.g., a humancell. In some embodiments, a first mRNA polynucleotide encodes a firstpolypeptide that is a heavy chain of an anti-CHIKV antibody, of aportion thereof (e.g., a heavy chain variable region), and a second mRNApolynucleotide encodes a second polypeptide that is a light chain of ananti-CHIKV antibody, or a portion thereof (e.g., a light chain variableregion), such that the first and second polynucleotides express theheavy and light chains of an anti-CHIKV antibody in a mammalian cell,e.g., a human cell, and the heavy and light chains pair to form theanti-CHIKV antibody.

In some embodiments, the anti-CHIKV antibody expressed in the cell issecreted and can bind to CHIKV and/or neutralize CHIKV. Anti-CHIKVantibody protein expression levels and/or anti-CHIKV antibody activity,e.g., antigen binding activity and/or virus neutralization activity, canbe measured according to methods known in the art. In some embodiments,the polynucleotide is introduced to the cells in vitro. In someembodiments, the polynucleotide is introduced to the cells in vivo byadministration of the polynucleotide to a subject.

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) that encodesa heavy chain polypeptide, e.g., SEQ ID NO: 1. In some embodiments, thepolynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise anucleotide sequence (e.g., an ORF) that encodes a light chainpolypeptide, e.g., SEQ ID NO: 3.

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) that isidentical to SEQ ID NO:2 or SEQ ID NO:4.

In some embodiments, the polynucleotides (e.g., an mRNA) describedherein comprise a nucleotide sequence that is identical to SEQ ID NO:5or SEQ ID NO: 6

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) that encodesa portion or a fragment of a heavy chain polypeptide or a light chainpolypeptide of an anti-CHIKV antibody. In some embodiments, thepolynucleotides (e.g., a RNA, e.g., an mRNA) of the invention comprise anucleotide sequence (e.g., an ORF) that encodes a heavy chain variableregion of a heavy chain antibody sequence polypeptide, e.g., amino acids21-142 of SEQ ID NO:1. In some embodiments, the polynucleotides (e.g., aRNA, e.g., an mRNA) of the invention comprise a nucleotide sequence(e.g., an ORF) that encodes a light chain variable region of a lightchain antibody sequence polypeptide, e.g., amino acids 21-128 of SEQ IDNO:3. In some embodiments, the polynucleotides (e.g., a RNA, e.g., anmRNA) of the invention comprise a nucleotide sequence (e.g., an ORF)that is identical to nucleotides 61-426 of SEQ ID NO:2, or nucleotides61-384 of SEQ ID NO:4.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a codon optimized nucleic acid sequence, whereinthe open reading frame (ORF) of the codon optimized nucleic acidsequence is derived from polypeptide of an anti-CHIKV antibody, e.g., aheavy chain polypeptide or a light chain polypeptide. For example, thepolynucleotides of invention can comprise a sequence optimized ORFencoding a heavy chain or a light chain of an anti-CHIKV antibody. Insome embodiments, the polynucleotides of the invention can comprise asequence optimized functional fragment of a heavy chain or light chainof an anti-CHIKV antibody, e.g., a variable region of a heavy chain orlight chain.

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) encoding amutant polypeptide of an anti-CHIKV antibody relative to a referenceanti-CHIKV antibody polypeptide. In some embodiments, thepolynucleotides of the invention comprise an ORF encoding an anti-CHIKVantibody polypeptide that comprises at least one point mutation in thepolypeptide sequence relative to a reference polypeptide, and retainsantigen binding activity and/or virus neutralization activity of thereference polypeptide. For example, the polynucleotides can comprise anORF encoding a heavy chain of an anti-CHIKV antibody with at least onepoint mutation relative to the heavy chain encoded by SEQ ID NO: 1. Forexample, the polynucleotides can comprise an ORF encoding a light chainof an anti-CHIKV antibody with at least one point mutation relative tothe light chain encoded by SEQ ID NO: 3. In some embodiments, ananti-CHIKV antibody having a mutant heavy chain and/or a mutant lightchain has an CHIKV neutralization activity which is at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 35%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, or at least 100% of the CHIKV neutralizationactivity of a corresponding anti-CHIKV antibody made up of the heavy andlight chains of SEQ ID NOs: 1 and 3 In some embodiments, an anti-CHIKVantibody having a mutant heavy chain and/or a mutant light chain has anCHIKV binding activity which is at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 100% of the CHIKV binding activity of a correspondinganti-CHIKV antibody made up of the heavy and light chains of SEQ ID NOs:1 and 3. In some embodiments, the polynucleotide (e.g., a RNA, e.g., anmRNA) of the invention comprising an ORF encoding a mutant anti-CHIKVantibody polypeptide is sequence optimized.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) thatencodes an anti-CHIKV antibody polypeptide with mutations that do notalter CHIKV binding and/or neutralization activity relative to ananti-CHIKV antibody comprising the heavy and light chains of SEQ ID NOs:1 and 3. Such mutant polypeptides can be referred to asfunction-neutral. In some embodiments, the polynucleotide comprises anORF that encodes a mutant anti-CHIKV antibody polypeptide comprising oneor more function-neutral point mutations.

In some embodiments, the anti-CHIKV antibody having a mutant heavy chainand/or light chain polypeptide has higher CHIKV binding and/orneutralization activity than the corresponding anti-CHIKV antibodyhaving the heavy and light chains of SEQ ID NOs: 1 and 3. In someembodiments, an anti-CHIKV antibody having a mutant heavy chain and/or amutant light chain has a CHIKV binding and/or neutralization activitywhich is at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or at least 100%higher than the CHIKV binding and/or neutralization activity of acorresponding anti-CHIKV antibody made up of the heavy and light chainsof SEQ ID NOs: 1 and 3.

In some embodiments, the polynucleotides (e.g., a RNA, e.g., an mRNA) ofthe invention comprise a nucleotide sequence (e.g., an ORF) encoding afunctional fragment of an anti-CHIKV antibody polypeptide, e.g., afunctional fragment of a heavy chain polypeptide or a light chainpolypeptide, such that the functional fragment of the polypeptide can,as part of an antibody, or antigen-binding portion thereof, bind toCHIKV and/or neutralize CHIKV. In some embodiments, an anti-CHIKVantibody, or antigen-binding portion thereof, having a functionalfragment of the heavy and/or light chain has a CHIKV binding and/orneutralization activity which is at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 100% of the CHIKV binding and/or neutralizationactivity of an anti-CHIKV antibody made up of the heavy and light chainsof SEQ ID NOs: 1 and 3. In some embodiments, an anti-CHIKV antibody, orantigen-binding portion thereof, having a functional fragment of theheavy and/or light chain has a CHIKV binding and/or neutralizationactivity which is at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least100% higher than the CHIKV binding and/or neutralization activity of ananti-CHIKV antibody made up of the heavy and light chains of SEQ ID NOs:1 and 3. In some embodiments, the polynucleotides (e.g., a RNA, e.g., anmRNA) of the invention comprising an ORF encoding a functional fragmentof an anti-CHIKV antibody polypeptide is sequence optimized.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding ananti-CHIKV antibody polypeptide fragment that is at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%,19%, 20%, 21%, 22%, 23%, 24% or 25% shorter than a corresponding fulllength anti-CHIKV antibody polypeptide, e.g., a full length heavy chainor a full length light chain of an anti-CHIKV antibody.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding ananti-CHIKV antibody heavy chain (e.g., a full length heavy chain,functional fragment of a heavy chain, or variant thereof), wherein thenucleotide sequence is at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% identical to the sequence of SEQ IDNO:2. In some embodiments, the polynucleotide (e.g., a RNA, e.g., anmRNA) of the invention comprises a nucleotide sequence (e.g., an ORF)encoding an anti-CHIKV antibody light chain (e.g., a full length lightchain, functional fragment of a light chain, or variant thereof),wherein the nucleotide sequence is at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100% identical to the sequence ofSEQ ID NO:4.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises an ORF encoding an anti-CHIKV antibodypolypeptide, wherein the polynucleotide comprises a nucleic acidsequence having 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 70%to 95%, 80% to 95%, 70% to 85%, 75% to 90%, 80% to 95%, 70% to 75%, 75%to 80%, 80% to 85%, 85% to 90%, 90% to 95%, or 95% to 100%, sequenceidentity to SEQ ID NO:2 or SEQ ID NO:4.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding ananti-CHIKV antibody polypeptide, wherein the nucleotide sequence differsfrom SEQ ID NO:2 or SEQ ID NO:4 by no more than 100 nucleotides, e.g.,by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100nucleotides.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding apolypeptide comprising the heavy chain variable region of the heavychain antibody sequence of SEQ ID NO:1, wherein the ORF has a nucleotidesequence that is at least 80% identical to nucleotides 61-426 of SEQ IDNO:2, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleotides61-426 of SEQ ID NO:2. In some embodiments, the polynucleotide (e.g., aRNA, e.g., an mRNA) of the invention comprises a nucleotide sequence(e.g., an ORF) encoding a polypeptide comprising the heavy chainvariable region of the heavy chain antibody sequence of SEQ ID NO:1,wherein the ORF comprises a nucleic acid sequence that differs from thenucleic acid sequence of nucleotides 61-426 of SEQ ID NO:2 by no morethan 75 nucleotides, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nucleotides.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleotide sequence (e.g., an ORF) encoding apolypeptide comprising the light chain variable region of the lightchain antibody sequence of SEQ ID NO:3, wherein the ORF has a nucleotidesequence that is at least 80% identical to nucleotides 61-384 of SEQ IDNO:4, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleotides61-384 of SEQ ID NO:4. In some embodiments, the polynucleotide (e.g., aRNA, e.g., an mRNA) of the invention comprises a nucleotide sequence(e.g., an ORF) encoding a polypeptide comprising the light chainvariable region of the light chain antibody sequence of SEQ ID NO:3,wherein the ORF comprises a nucleic acid sequence that differs from thenucleic acid sequence of nucleotides 61-384 of SEQ ID NO:4 by no morethan 75 nucleotides, e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 nucleotides.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleic acid sequence that is at least 80%identical to SEQ ID NO:2, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO:2. In some embodiments, the polynucleotide (e.g.,a RNA, e.g., an mRNA) of the invention comprises a nucleic acid sequencethat is at least 80% identical to nucleotides 61-1416 of SEQ ID NO:2,e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleotides61-1416 of SEQ ID NO:2.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises a nucleic acid sequence that is at least 80%identical to SEQ ID NO:4, e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to SEQ ID NO:4. In some embodiments, polynucleotide (e.g., aRNA, e.g., an mRNA) of the invention comprises a nucleic acid sequencethat is at least 80% identical to nucleotides 61-705 of SEQ ID NO:4,e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to nucleotides61-705 of SEQ ID NO:4.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe invention comprises from about 30 to about 100,000 nucleotides(e.g., from 30 to 100, from 30 to 200, from 50 to 300, from 100 to 400,from 200 to 500, from 200 to 600, from 300 to 700, from 400 to 800, from500 to 900, from 900 to 1,000, from 900 to 1,100, from 900 to 1,200,from 900 to 1,300, from 900 to 1,400, from 900 to 1,500, from 1,000 to1,100, from 1,000 to 1,100, from 1,000 to 1,200, from 1,000 to 1,300,from 1,000 to 1,400, from 1,000 to 1,500, from 1,187 to 1,200, from1,187 to 1,400, from 1,187 to 1,600, from 1,187 to 1,800, from 1,187 to2,000, from 1,187 to 3,000, from 1,187 to 5,000, from 1,187 to 7,000,from 1,187 to 10,000, from 1,187 to 25,000, from 1,187 to 50,000, from1,187 to 70,000, or from 1,187 to 100,000).

In some embodiments, a polynucleotide (e.g., a RNA, e.g., an mRNA)described herein comprises a nucleotide sequence (e.g., an ORF) encodinga polypeptide, wherein the length of the nucleotide sequence (e.g., anORF) is at least 30 nucleotides in length (e.g., at least or greaterthan about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160,180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000,1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000,2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000,20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up toand including 100,000 nucleotides).

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprising a nucleotide sequence (e.g., an ORF) encodingan anti-CHIKV antibody polypeptide (e.g., a heavy chain polypeptide orlight chain polypeptide of an anti-CHIKV antibody, fragments thereof, orvariants thereof) further comprises at least one nucleic acid sequencethat is noncoding, e.g., a microRNA binding site. In some embodiments,the polynucleotide (e.g., a RNA, e.g., an mRNA) of the invention furthercomprises a 5′-UTR (e.g., selected from the sequences of SEQ ID NOs:13and 108-126) and a 3′UTR (e.g., selected from the sequences of SEQ IDNOs:14 and 127-138). In some embodiments, the polynucleotide (e.g., aRNA, e.g., an mRNA) of the invention comprises a sequence selected fromSEQ ID NO:2 or SEQ ID NO:4. In a further embodiment, the polynucleotide(e.g., a RNA, e.g., an mRNA) comprises a 5′ terminal cap (e.g., Cap0,Cap1, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof) anda poly-A-tail region (e.g., about 100 nucleotides in length). In someembodiments, an mRNA described herein comprises a 5′ UTR comprising anucleic acid sequence of SEQ ID NO:13. In some embodiments, an mRNAdescribed herein comprises a 3′ UTR comprising a nucleic acid sequenceof SEQ ID NO:14. In some embodiments, the mRNA comprises a polyA tail.In some instances, the poly A tail is 50-150, 75-150, 85-150, 90-150,90-120, 90-130, or 90-150 nucleotides in length. In some instances, thepoly A tail is 100 nucleotides in length.

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprising a nucleotide sequence (e.g., an ORF) encodingan anti-CHIKV antibody polypeptide is single stranded or doublestranded.

In some embodiments, the polynucleotide of the invention comprising anucleotide sequence (e.g., an ORF) encoding an anti-CHIKV antibodypolypeptide is DNA or RNA. In some embodiments, the polynucleotide ofthe invention is RNA. In some embodiments, the polynucleotide of theinvention is, or functions as, an mRNA. In some embodiments, the mRNAcomprises a nucleotide sequence (e.g., an ORF) that encodes at least oneanti-CHIKV antibody polypeptide, and is capable of being translated toproduce the encoded anti-CHIKV antibody polypeptide in vitro, in vivo,in situ or ex vivo.

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) comprises a sequence-optimized nucleotide sequence (e.g.,an ORF) encoding an anti-CHIKV antibody polypeptide (e.g., SEQ ID NOs:2or 4), wherein the polynucleotide comprises at least one chemicallymodified nucleobase, e.g., N1-methylpseudouracil or 5-methoxyuracil. Incertain embodiments, all uracils in the polynucleotide areN1-methylpseudouracils. In other embodiments, all uracils in thepolynucleotide are 5-methoxyuracils. In some embodiments, thepolynucleotide further comprises a miRNA binding site, e.g., a miRNAbinding site that binds to miR-142 and/or a miRNA binding site thatbinds to miR-126.

In some embodiments, the polynucleotide (e.g., a RNA, e.g., a mRNA)disclosed herein is formulated with a delivery agent comprising, e.g., acompound having the Formula (I), e.g., any of Compounds 1-232, e.g.,Compound II; a compound having the Formula (III), (IV), (V), or (VI),e.g., any of Compounds 233-342, e.g., Compound VI; or a compound havingthe Formula (VIII), e.g., any of Compounds 419-428, e.g., Compound I, orany combination thereof. In some embodiments, the delivery agentcomprises Compound II, DSPC, Cholesterol, and Compound I or PEG-DMG,e.g., with a mole ratio of about 50:10:38.5:1.5. In some embodiments,the delivery agent comprises Compound VI, DSPC, Cholesterol, andCompound I or PEG-DMG, e.g., with a mole ratio in the range of about 30to about 60 mol % Compound II or VI (or related suitable amino lipid)(e.g., 30-40, 40-45, 45-50, 50-55 or 55-60 mol % Compound II or VI (orrelated suitable amino lipid)), about 5 to about 20 mol % phospholipid(or related suitable phospholipid or “helper lipid”) (e.g., 5-10, 10-15,or 15-20 mol % phospholipid (or related suitable phospholipid or “helperlipid”)), about 20 to about 50 mol % cholesterol (or related sterol or“non-cationic” lipid) (e.g., about 20-30, 30-35, 35-40, 40-45, or 45-50mol % cholesterol (or related sterol or “non-cationic” lipid)) and about0.05 to about 10 mol % PEG lipid (or other suitable PEG lipid) (e.g.,0.05-1, 1-2, 2-3, 3-4, 4-5, 5-7, or 7-10 mol % PEG lipid (or othersuitable PEG lipid)). An exemplary delivery agent can comprise moleratios of, for example, 47.5:10.5:39.0:3.0 or 50:10:38.5:1.5. In certaininstances, an exemplary delivery agent can comprise mole ratios of, forexample, 47.5:10.5:39.0:3; 47.5:10:39.5:3; 47.5:11:39.5:2;47.5:10.5:39.5:2.5; 47.5:11:39:2.5; 48.5:10:38.5:3; 48.5:10.5:39:2;48.5:10.5:38.5:2.5; 48.5:10.5:39.5:1.5; 48.5:10.5:38.0:3;47:10.5:39.5:3; 47:10:40.5:2.5; 47:11:40:2; 47:10.5:39.5:3;48:10.5:38.5:3; 48:10:39.5:2.5; 48:11:39:2; or 48:10.5:38.5:3. In someembodiments, the delivery agent comprises Compound II or VI, DSPC,Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about47.5:10.5:39.0:3.0. In some embodiments, the delivery agent comprisesCompound II or VI, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g.,with a mole ratio of about 50:10:38.5:1.5. In some embodiments, thedelivery agent comprises Compound II, DSPC, Cholesterol, and Compound I,e.g., with a mole ratio of about 50:10:38:2. In certain instances, anexemplary delivery agent can comprise a mole ratio of about 50:10:38:2.In some embodiments, the delivery agent comprises Compound II, DSPC,Cholesterol, and Compound I, e.g., with a mole ratio in the range ofabout 30 to about 60 mol % Compound II (or related suitable amino lipid)(e.g., 30-40, 40-45, 45-50, 50-55 or 55-60 mol % Compound II (or relatedsuitable amino lipid)), about 5 to about 20 mol % phospholipid (orrelated suitable phospholipid or “helper lipid”) (e.g., 5-10, 10-15, or15-20 mol % phospholipid (or related suitable phospholipid or “helperlipid”)), about 20 to about 50 mol % cholesterol (or related sterol or“non-cationic” lipid) (e.g., about 20-30, 30-35, 35-40, 40-45, or 45-50mol % cholesterol (or related sterol or “non-cationic” lipid)) and about0.05 to about 10 mol % Compound I (or other suitable PEG lipid) (e.g.,0.05-1, 1-2, 2-3, 3-4, 4-5, 5-7, or 7-10 mol % PEG lipid (or othersuitable PEG lipid)).

In some embodiments, the polynucleotide of the disclosure is an mRNAthat comprises a 5′-terminal cap (e.g., Cap 1), a 5′UTR (e.g., SEQ IDNO:13), a ORF sequence selected from the group consisting of SEQ IDNOs.:2 and 4, a 3′UTR (e.g., SEQ ID NO:14), and a poly A tail (e.g.,about 100 nucleotides in length), wherein all uracils in thepolynucleotide are N1-methylpseudouracils. In some embodiments, thedelivery agent comprises Compound II or Compound VI as the ionizablelipid and PEG-DMG or Compound I as the PEG lipid, or any combinationsthereof (e.g., Compound I and Compound II). In some embodiments, thedelivery agent comprises Compound II, DSPC, cholesterol, and Compound I.

Treatments for Chikungunya virus infection, as provided herein, compriseat least one (e.g., one or more) RNA (e.g., mRNA) polynucleotide havingan open reading frame encoding at least one antibody, antibody domain,antibody portion, and/or antibody fragment thereof, wherein theantibody, antibody portion, or antibody fragment binds to Chikungunyavirus, or wherein two or more antibody portions or fragments associateto form an antibody, or antigen-binding portion thereof, that binds tochikungunya virus. The terms “polynucleotide” and “nucleic acid,” intheir broadest sense, include any compound and/or substance thatcomprises a polymer of nucleotides. Polynucleotides (also referred to asnucleic acids) may be or may include, for example, RNAs,deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycolnucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids(LNAs, including LNA having a β-D-ribo configuration, α-LNA having anα-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a2′-amino functionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleicacids (CeNA) or chimeras or combinations thereof.

In some embodiments, an RNA polynucleotide encodes 1-10, 2-10, 2-9, 2-8,2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9,4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10,7-9, 7-8, 8-10, 8-9 or 9-10 antibodies, antibody fragments, or antigenbinding fragments.

3. Signal Sequences

The polynucleotides (e.g., a RNA, e.g., an mRNA) of the invention canalso comprise nucleotide sequences that encode additional features thatfacilitate trafficking of the encoded polypeptides to therapeuticallyrelevant sites. One such feature that aids in protein trafficking is thesignal sequence, or targeting sequence. The peptides encoded by thesesignal sequences are known by a variety of names, including targetingpeptides, transit peptides, and signal peptides. In some embodiments,the polynucleotide (e.g., a RNA, e.g., an mRNA) comprises a nucleotidesequence (e.g., an ORF) that encodes a signal peptide operably linked toa nucleotide sequence that encodes anti-CHIKV antibody polypeptidedescribed herein.

In some embodiments, the “signal sequence” or “signal peptide” is apolynucleotide or polypeptide, respectively, which is from about 30-210nucleotides, e.g., about 45-80 or 15-60 nucleotides (e.g., about 20, 30,40, 50, 60, or 70 amino acids) in length that, optionally, isincorporated at the 5′ (or N-terminus) of the coding region or thepolypeptide, respectively. Addition of these sequences results intrafficking the encoded polypeptide to a desired site, such as theendoplasmic reticulum or the mitochondria through one or more targetingpathways. Some signal peptides are cleaved from the protein, for exampleby a signal peptidase after the proteins are transported to the desiredsite.

In some embodiments, the polynucleotide of the present disclosurecomprises a nucleotide sequence encoding an anti-CHIKV antibodypolypeptide (e.g., a heavy chain polypeptide or a light chainpolypeptide), wherein the nucleotide sequence further comprises a 5′nucleic acid sequence encoding a native signal peptide. In anotherembodiment, the polynucleotide of the present disclosure comprises anucleotide sequence encoding an anti-CHIKV antibody polypeptide (e.g., aheavy chain polypeptide or a light chain polypeptide), wherein thenucleotide sequence lacks the nucleic acid sequence encoding a nativesignal peptide.

In some embodiments, the polynucleotide of the present disclosurecomprises a nucleotide sequence encoding an anti-CHIKV antibodypolypeptide (e.g., a heavy chain polypeptide or a light chainpolypeptide), wherein the nucleotide sequence further comprises a 5′nucleic acid sequence encoding a heterologous signal peptide.

4. Fusion Proteins

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) can comprise more than one nucleic acid sequence (e.g.,an ORF) encoding a polypeptide of interest. In some embodiments,polynucleotides of the invention comprise a single ORF encoding ananti-CHIKV antibody polypeptide, a functional fragment, or a variantthereof. However, in some embodiments, the polynucleotide of theinvention can comprise more than one ORF, for example, a first ORFencoding an anti-CHIKV antibody polypeptide (a first polypeptide ofinterest), a functional fragment, or a variant thereof, and a second ORFexpressing a second polypeptide of interest. In some embodiments, two ormore polypeptides of interest can be genetically fused, i.e., two ormore polypeptides can be encoded by the same ORF. In some embodiments,the polynucleotide can comprise a nucleic acid sequence encoding alinker (e.g., a G4S (SEQ ID NO:230)) peptide linker or another linkerknown in the art) between two or more polypeptides of interest.

In some embodiments, a polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) can comprise two, three, four, or more ORFs, eachexpressing a polypeptide of interest.

In some embodiments, the polynucleotide of the invention (e.g., a RNA,e.g., an mRNA) can comprise a first nucleic acid sequence (e.g., a firstORF) encoding an anti-CHIKV antibody polypeptide and a second nucleicacid sequence (e.g., a second ORF) encoding a second polypeptide ofinterest, e.g., a second anti-CHIKV antibody polypeptide.

Linkers and Cleavable Peptides

In certain embodiments, the mRNAs of the disclosure encode more than oneanti-CHIKV antibody polypeptide (e.g., an antibody heavy chain and anantibody light chain) or a heterologous polypeptide, referred to hereinas multimer constructs. In certain embodiments of the multimerconstructs, the mRNA further encodes a linker located between eachpolypeptide. The linker can be, for example, a cleavable linker orprotease-sensitive linker. In certain embodiments, the linker isselected from the group consisting of F2A linker, P2A linker, T2Alinker, E2A linker, and combinations thereof. This family ofself-cleaving peptide linkers, referred to as 2A peptides, has beendescribed in the art (see for example, Kim, J. H. et al. (2011) PLoS ONE6:e18556). In certain embodiments, the linker is an F2A linker. Incertain embodiments, the linker is a GGGS linker. In certainembodiments, the linker is a (GGGS)n linker, wherein n=2, 3, 4, or 5. Incertain embodiments, the multimer construct contains two or morepolypeptides with intervening linkers, having the structure:polypeptide-linker-polypeptide-linker-polypeptide.

In one embodiment, the cleavable linker is an F2A linker (e.g., havingthe amino acid sequence GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO:223)). Inother embodiments, the cleavable linker is a T2A linker (e.g., havingthe amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO:224)), a P2Alinker (e.g., having the amino acid sequence GSGATNFSLLKQAGDVEENPGP (SEQID NO:225)) or an E2A linker (e.g., having the amino acid sequenceGSGQCTNYALLKLAGDVESNPGP (SEQ ID NO:226)). The skilled artisan willappreciate that other art-recognized linkers may be suitable for use inthe constructs of the invention (e.g., encoded by the polynucleotides ofthe invention). The skilled artisan will likewise appreciate that othermulticistronic constructs may be suitable for use in the invention. Inexemplary embodiments, the construct design yields approximatelyequimolar amounts of intrabody and/or domain thereof encoded by theconstructs of the invention.

In one embodiment, the self-cleaving peptide may be, but is not limitedto, a 2A peptide. A variety of 2A peptides are known and available inthe art and may be used, including e.g., the foot and mouth diseasevirus (FMDV) 2A peptide, the equine rhinitis A virus 2A peptide, theThosea asigna virus 2A peptide, and the porcine teschovirus-1 2Apeptide. 2A peptides are used by several viruses to generate twoproteins from one transcript by ribosome-skipping, such that a normalpeptide bond is impaired at the 2A peptide sequence, resulting in twodiscontinuous proteins being produced from one translation event. In oneembodiment, the 2A peptide cleaves between the last glycine and lastproline. One example of a polynucleotide sequence encoding the 2Apeptide is: GGAAGCGGAGCUACUAACUUCAGCCUGCUGAAGCAGGCUGGAGACGUGGAGGAGAACCCUGGACCU (SEQ ID NO:227). In one illustrative embodiment, a 2Apeptide is encoded by the following sequence: 5′UCCGGACUCAGAUCCGGGGAUCUCAAAAUUGUCGCUCCUGUCAAACAAACUCUUAACUUUGAUUUACUCAAACUGGCTGGGGAUGUAGAAAGCAAUCCAGGTCCAC UC-3′ (SEQ IDNO:228). The polynucleotide sequence of the 2A peptide may be modifiedor codon optimized by the methods described herein and/or are known inthe art.

In one embodiment, this sequence may be used to separate the codingregions of two or more polypeptides of interest. As a non-limitingexample, the sequence encoding the F2A peptide may be between a firstcoding region A and a second coding region B (A-F2Apep-B). The presenceof the F2A peptide results in the cleavage of the one long proteinbetween the glycine and the proline at the end of the F2A peptidesequence (NPGP is cleaved to result in NPG and P) thus creating separateprotein A (with 21 amino acids of the F2A peptide attached, ending withNPG) and separate protein B (with 1 amino acid, P, of the F2A peptideattached). Likewise, for other 2A peptides (P2A, T2A and E2A), thepresence of the peptide in a long protein results in cleavage betweenthe glycine and proline at the end of the 2A peptide sequence (NPGP iscleaved to result in NPG and P). Protein A and protein B may be the sameor different peptides or polypeptides of interest (e.g., an anti-CHIKVantibody heavy chain and an anti-CHIKV antibody light chain, orfragments thereof). In particular embodiments, protein A and protein Bare anti-CHIKV antibody heavy and light chains, in either order.

5. Sequence Optimization of Nucleotide Sequence Encoding an AntibodyPolypeptide

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe present disclosure is sequence optimized. In some embodiments, thepolynucleotide (e.g., a RNA, e.g., an mRNA) of the present disclosurecomprises a nucleotide sequence (e.g., an ORF) encoding an anti-CHIKVantibody polypeptide, optionally, a nucleotide sequence (e.g., an ORF)encoding another polypeptide of interest, a 5′-UTR, a 3′-UTR, the 5′ UTRor 3′ UTR optionally comprising at least one microRNA binding site,optionally a nucleotide sequence encoding a linker, a polyA tail, or anycombination thereof), in which the ORF(s) that are sequence optimized.

A sequence-optimized nucleotide sequence, e.g., a codon-optimized mRNAsequence encoding an anti-CHIKV antibody polypeptide, is a sequencecomprising at least one synonymous nucleobase substitution with respectto a reference sequence (e.g., a nucleotide sequence encoding areference anti-CHIKV antibody polypeptide).

A sequence-optimized nucleotide sequence can be partially or completelydifferent in sequence from the reference sequence. For example, areference sequence encoding polyserine uniformly encoded by UCU codonscan be sequence-optimized by having 100% of its nucleobases substituted(for each codon, U in position 1 replaced by A, C in position 2 replacedby G, and U in position 3 replaced by C) to yield a sequence encodingpolyserine which would be uniformly encoded by AGC codons. Thepercentage of sequence identity obtained from a global pairwisealignment between the reference polyserine nucleic acid sequence and thesequence-optimized polyserine nucleic acid sequence would be 0%.However, the protein products from both sequences would be 100%identical.

Some sequence optimization (also sometimes referred to codonoptimization) methods are known in the art (and discussed in more detailbelow) and can be useful to achieve one or more desired results. Theseresults can include, e.g., matching codon frequencies in certain tissuetargets and/or host organisms to ensure proper folding; biasing G/Ccontent to increase mRNA stability or reduce secondary structures;minimizing tandem repeat codons or base runs that can impair geneconstruction or expression; customizing transcriptional andtranslational control regions; inserting or removing protein traffickingsequences; removing/adding post translation modification sites in anencoded protein (e.g., glycosylation sites); adding, removing orshuffling protein domains; inserting or deleting restriction sites;modifying ribosome binding sites and mRNA degradation sites; adjustingtranslational rates to allow the various domains of the protein to foldproperly; and/or reducing or eliminating problem secondary structureswithin the polynucleotide. Sequence optimization tools, algorithms andservices are known in the art, non-limiting examples include servicesfrom GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/orproprietary methods.

Codon options for each amino acid are given in the following TABLE.

Single Letter Amino Acid Code Codon Options Isoleucine I AUU, AUC, AUALeucine L CUU, CUC, CUA, CUG, UUA, UUG Valine V GUU, GUC, GUA, GUGPhenylalanine F UUU, UUC Methionine M AUG Cysteine C UGU, UGC Alanine AGCU, GCC, GCA, GCG Glycine G GGU, GGC, GGA, GGG Proline P CCU, CCC, CCA,CCG Threonine T ACU, ACC, ACA, ACG Serine S UCU, UCC, UCA, UCG, AGU, AGCTyrosine Y UAU, UAC Tryptophan W UGG Glutamine Q CAA, CAG Asparagine NAAU, AAC Histidine H CAU, CAC Glutamic acid E GAA, GAG Aspartic acid DGAU, GAC Lysine K AAA, AAG Arginine R CGU, CGC, CGA, CGG, AGA, AGGSelenocysteine Sec UGA in mRNA in presence of Selenocysteine insertionelement (SECIS) Stop codons Stop UAA, UAG, UGA

In some embodiments, a polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe present disclosure comprises a sequence-optimized nucleotidesequence (e.g., an ORF) encoding an anti-CHIKV antibody polypeptide, afunctional fragment, or a variant thereof, wherein the antibodypolypeptide, functional fragment, or a variant thereof encoded by thesequence-optimized nucleotide sequence has improved properties (e.g.,compared to an anti-CHIKV antibody polypeptide, functional fragment, ora variant thereof encoded by a reference nucleotide sequence that is notsequence optimized), e.g., improved properties related to expressionefficacy after administration in vivo. Such properties include, but arenot limited to, improving nucleic acid stability (e.g., mRNA stability),increasing translation efficacy in the target tissue, reducing thenumber of truncated proteins expressed, improving the folding or preventmisfolding of the expressed proteins, reducing toxicity of the expressedproducts, reducing cell death caused by the expressed products,increasing and/or decreasing protein aggregation.

In some embodiments, the sequence-optimized nucleotide sequence (e.g.,an ORF) is codon optimized for expression in human subjects, havingstructural and/or chemical features that avoid one or more of theproblems in the art, for example, features which are useful foroptimizing formulation and delivery of nucleic acid-based therapeuticswhile retaining structural and functional integrity; overcoming athreshold of expression; improving expression rates; half-life and/orprotein concentrations; optimizing protein localization; and avoidingdeleterious bio-responses such as the immune response and/or degradationpathways.

In some embodiments, the polynucleotides of the invention comprise anucleotide sequence (e.g., a nucleotide sequence (e.g., an ORF) encodingan anti-CHIKV antibody polypeptide, a nucleotide sequence (e.g., an ORF)encoding another polypeptide of interest, a 5′-UTR, a 3′-UTR, a microRNAbinding site, a nucleic acid sequence encoding a linker, or anycombination thereof) that is sequence-optimized according to a methodcomprising:

-   -   (i) substituting at least one codon in a reference nucleotide        sequence (e.g., an ORF encoding an anti-CHIKV antibody        polypeptide) with an alternative codon to increase or decrease        uridine content to generate a uridine-modified sequence;    -   (ii) substituting at least one codon in a reference nucleotide        sequence (e.g., an ORF encoding an anti-CHIKV antibody        polypeptide) with an alternative codon having a higher codon        frequency in the synonymous codon set;    -   (iii) substituting at least one codon in a reference nucleotide        sequence (e.g., an ORF encoding an anti-CHIKV antibody        polypeptide) with an alternative codon to increase G/C content;        or    -   (iv) a combination thereof.

In some embodiments, the sequence-optimized nucleotide sequence (e.g.,an ORF encoding an anti-CHIKV antibody polypeptide) has at least oneimproved property with respect to the reference nucleotide sequence.

In some embodiments, the sequence optimization method is multiparametricand comprises one, two, three, four, or more methods disclosed hereinand/or other optimization methods known in the art.

Features, which can be considered beneficial in some embodiments of thepresent disclosure, can be encoded by or within regions of thepolynucleotide and such regions can be upstream (5′) to, downstream (3′)to, or within the region that encodes the anti-CHIKV antibodypolypeptide. These regions can be incorporated into the polynucleotidebefore and/or after sequence-optimization of the protein encoding regionor open reading frame (ORF). Examples of such features include, but arenot limited to, untranslated regions (UTRs), microRNA sequences, Kozaksequences, oligo(dT) sequences, poly-A tail, and detectable tags and caninclude multiple cloning sites that can have XbaI recognition.

In some embodiments, the polynucleotide of the present disclosurecomprises a 5′ UTR. a 3′ UTR and/or a miRNA binding site. In someembodiments, the polynucleotide comprises two or more 5′ UTRs and/or 3′UTRs, which can be the same or different sequences. In some embodiments,the polynucleotide comprises two or more miRNA binding sites, which canbe the same or different sequences. Any portion of the 5′ UTR, 3′ UTR,and/or miRNA binding site, including none, can be sequence-optimized andcan independently contain one or more different structural or chemicalmodifications, before and/or after sequence optimization.

In some embodiments, after optimization, the polynucleotide isreconstituted and transformed into a vector such as, but not limited to,plasmids, viruses, cosmids, and artificial chromosomes. For example, theoptimized polynucleotide can be reconstituted and transformed intochemically competent E. coli, yeast, neurospora, maize, drosophila, etc.where high copy plasmid-like or chromosome structures occur by methodsdescribed herein.

Exemplary amino acid sequences and nucleotide sequences encoding humananti-CHIKV antibody polypeptides are provided in the Construct SequencesAppendix.

6. Sequence-Optimized Nucleotide Sequences Encoding AntibodyPolypeptides

In some embodiments, the polynucleotide described herein comprises asequence-optimized nucleotide sequence encoding an anti-CHIKV antibodypolypeptide disclosed herein. In some embodiments, the polynucleotide ofthe present disclosure comprises an open reading frame (ORF) encoding ananti-CHIKV antibody polypeptide, wherein the ORF has been sequenceoptimized.

Exemplary sequence-optimized nucleotide sequences encoding humananti-CHIKV antibody polypeptides are set forth as SEQ ID NOs: 2 and 4(CHIKV24 heavy chain and CHIKV24 light chain, respectively). In someembodiments, the sequence optimized anti-CHIKV antibody sequences,fragments, and variants thereof are used to practice the methodsdisclosed herein.

In some embodiments, a polynucleotide of the present disclosure, forexample a polynucleotide comprising an mRNA nucleotide sequence encodingan anti-CHIKV antibody polypeptide, comprises from 5′ to 3′ end:

-   -   (i) a 5′ cap provided herein, for example, Cap1;    -   (ii) a 5′ UTR, such as the sequences provided herein, for        example, SEQ ID NO: 13;    -   (iii) an open reading frame encoding an anti-CHIKV antibody        polypeptide, e.g., a sequence optimized nucleic acid sequence        encoding an anti-CHIKV antibody polypeptide set forth as SEQ ID        NO:2 or SEQ ID NO:4, or a fragment thereof (e.g., a heavy chain        variable region or a light chain variable region);    -   (iv) at least one stop codon;    -   (v) a 3′ UTR, such as the sequences provided herein, for        example, SEQ ID NO: 14; and    -   (vi) a poly-A tail provided above.

In certain embodiments, all uracils in the polynucleotide are N1methylpseudouracils (G5). In certain embodiments, all uracils in thepolynucleotide are 5-methoxyuracils (G6).

The sequence-optimized nucleotide sequences disclosed herein aredistinct from the corresponding wild type nucleotide acid sequences andfrom other known sequence-optimized nucleotide sequences, e.g., thesesequence-optimized nucleic acids have unique compositionalcharacteristics.

In some embodiments, the percentage of uracil or thymine nucleobases ina sequence-optimized nucleotide sequence (e.g., encoding an anti-CHIKVantibody polypeptide, a functional fragment, or a variant thereof) ismodified (e.g., reduced) with respect to the percentage of uracil orthymine nucleobases in the reference wild-type nucleotide sequence. Sucha sequence is referred to as a uracil-modified or thymine-modifiedsequence. The percentage of uracil or thymine content in a nucleotidesequence can be determined by dividing the number of uracils or thyminesin a sequence by the total number of nucleotides and multiplying by 100.In some embodiments, the sequence-optimized nucleotide sequence has alower uracil or thymine content than the uracil or thymine content inthe reference wild-type sequence. In some embodiments, the uracil orthymine content in a sequence-optimized nucleotide sequence of thepresent disclosure is greater than the uracil or thymine content in thereference wild-type sequence and still maintain beneficial effects,e.g., increased expression and/or reduced Toll-Like Receptor (TLR)response when compared to the reference wild-type sequence.

Methods for optimizing codon usage are known in the art. For example, anORF of any one or more of the sequences provided herein may be codonoptimized. Codon optimization, in some embodiments, may be used to matchcodon frequencies in target and host organisms to ensure proper folding;bias GC content to increase mRNA stability or reduce secondarystructures; minimize tandem repeat codons or base runs that may impairgene construction or expression; customize transcriptional andtranslational control regions; insert or remove protein traffickingsequences; remove/add post translation modification sites in encodedprotein (e.g., glycosylation sites); add, remove or shuffle proteindomains; insert or delete restriction sites; modify ribosome bindingsites and mRNA degradation sites; adjust translational rates to allowthe various domains of the protein to fold properly; or reduce oreliminate problem secondary structures within the polynucleotide. Codonoptimization tools, algorithms and services are known in theart—non-limiting examples include services from GeneArt (LifeTechnologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. Insome embodiments, the open reading frame (ORF) sequence is optimizedusing optimization algorithms.

7. Characterization of Sequence Optimized Nucleic Acids

In some embodiments of the present disclosure, the polynucleotide (e.g.,a RNA, e.g., an mRNA) comprising a sequence optimized nucleic aciddisclosed herein encoding an anti-CHIKV antibody polypeptide can be canbe tested to determine whether at least one nucleic acid sequenceproperty (e.g., stability when exposed to nucleases) or expressionproperty has been improved with respect to the non-sequence optimizednucleic acid.

As used herein, “expression property” refers to a property of a nucleicacid sequence either in vivo (e.g., translation efficacy of a syntheticmRNA after administration to a subject in need thereof) or in vitro(e.g., translation efficacy of a synthetic mRNA tested in an in vitromodel system). Expression properties include but are not limited to theamount of protein produced by an mRNA encoding an antibody afteradministration, and the amount of soluble or otherwise functionalprotein produced. In some embodiments, sequence optimized nucleic acidsdisclosed herein can be evaluated according to the viability of thecells expressing a protein encoded by a sequence optimized nucleic acidsequence (e.g., a RNA, e.g., an mRNA) encoding an anti-CHIKV antibodypolypeptide disclosed herein.

In a particular embodiment, a plurality of sequence optimized nucleicacids disclosed herein (e.g., a RNA, e.g., an mRNA) containing codonsubstitutions with respect to the non-optimized reference nucleic acidsequence can be characterized functionally to measure a property ofinterest, for example an expression property in an in vitro modelsystem, or in vivo in a target tissue or cell.

a. Optimization of Nucleic Acid Sequence Intrinsic Properties

In some embodiments of the present disclosure, the desired property ofthe polynucleotide is an intrinsic property of the nucleic acidsequence. For example, the nucleotide sequence (e.g., a RNA, e.g., anmRNA) can be sequence optimized for in vivo or in vitro stability. Insome embodiments, the nucleotide sequence can be sequence optimized forexpression in a particular target tissue or cell. In some embodiments,the nucleic acid sequence is sequence optimized to increase its plasmahalf by preventing its degradation by endo and exonucleases.

In other embodiments, the nucleic acid sequence is sequence optimized toincrease its resistance to hydrolysis in solution, for example, tolengthen the time that the sequence optimized nucleic acid or apharmaceutical composition comprising the sequence optimized nucleicacid can be stored under aqueous conditions with minimal degradation.

In other embodiments, the sequence optimized nucleic acid can beoptimized to increase its resistance to hydrolysis in dry storageconditions, for example, to lengthen the time that the sequenceoptimized nucleic acid can be stored after lyophilization with minimaldegradation.

b. Nucleic Acids Sequence Optimized for Protein Expression

In some embodiments of the present disclosure, the desired property ofthe polynucleotide is the level of expression of an antibody encoded bya sequence optimized sequence disclosed herein. Protein expressionlevels can be measured using one or more expression systems. In someembodiments, expression can be measured in cell culture systems, e.g.,CHO cells or HEK293 cells. In some embodiments, expression can bemeasured using in vitro expression systems prepared from extracts ofliving cells, e.g., rabbit reticulocyte lysates, or in vitro expressionsystems prepared by assembly of purified individual components. In otherembodiments, the protein expression is measured in an in vivo system,e.g., mouse, rabbit, monkey, etc.

In some embodiments, protein expression in solution form can bedesirable. Accordingly, in some embodiments, a reference sequence can besequence optimized to yield a sequence optimized nucleic acid sequencehaving optimized levels of expressed proteins in soluble form. Levels ofprotein expression and other properties such as solubility, levels ofaggregation, and the presence of truncation products (i.e., fragmentsdue to proteolysis, hydrolysis, or defective translation) can bemeasured according to methods known in the art, for example, usingelectrophoresis (e.g., native or SDS-PAGE) or chromatographic methods(e.g., HPLC, size exclusion chromatography, etc.).

c. Optimization of Target Tissue or Target Cell Viability

In some embodiments, the expression of heterologous therapeutic proteinsencoded by a nucleic acid sequence can have deleterious effects in thetarget tissue or cell, reducing protein yield, or reducing the qualityof the expressed product (e.g., due to the presence of protein fragmentsor precipitation of the expressed protein in inclusion bodies), orcausing toxicity.

Accordingly, in some embodiments of the present disclosure, the sequenceoptimization of a nucleic acid sequence disclosed herein, e.g., anucleic acid sequence encoding an anti-CHIKV antibody polypeptide, canbe used to increase the viability of target cells expressing the proteinencoded by the sequence optimized nucleic acid.

Heterologous protein expression can also be deleterious to cellstransfected with a nucleic acid sequence for autologous or heterologoustransplantation. Accordingly, in some embodiments of the presentdisclosure the sequence optimization of a nucleic acid sequencedisclosed herein can be used to increase the viability of target cellsexpressing the protein encoded by the sequence optimized nucleic acidsequence. Changes in cell or tissue viability, toxicity, and otherphysiological reaction can be measured according to methods known in theart.

d Reduction of Immune and/or Inflammatory Response

In some cases, the administration of a sequence optimized nucleic acidencoding an anti-CHIKV antibody polypeptide or a functional fragmentthereof can trigger an immune response, which could be caused by (i) thetherapeutic agent (e.g., an mRNA encoding an anti-CHIKV antibodypolypeptide), or (ii) the expression product of such therapeutic agent(e.g., the anti-CHIKV antibody polypeptide encoded by the mRNA), or (iv)a combination thereof. Accordingly, in some embodiments of the presentdisclosure the sequence optimization of nucleic acid sequence (e.g., anmRNA) disclosed herein can be used to decrease an immune or inflammatoryresponse (other than coagulation pathway activation) triggered by theadministration of a nucleic acid encoding an anti-CHIKV antibodypolypeptide or by the expression product of anti-CHIKV antibodypolypeptide encoded by such nucleic acid.

In some aspects, an inflammatory response can be measured by detectingincreased levels of one or more inflammatory cytokines using methodsknown in the art, e.g., ELISA. The term “inflammatory cytokine” refersto cytokines that are elevated in an inflammatory response. Examples ofinflammatory cytokines include interleukin-6 (IL-6), CXCL1 (chemokine(C—X—C motif) ligand 1; also known as GROα, interferon-γ (IFNγ), tumornecrosis factor α (TNFα), interferon γ-induced protein 10 (IP-10), orgranulocyte-colony stimulating factor (G-CSF). The term inflammatorycytokines also includes other cytokines associated with inflammatoryresponses known in the art, e.g., interleukin-1 (IL-1), interleukin-8(IL-8), interleukin-12 (IL-12), interleukin-13 (Il-13), interferon α(IFN-α), etc.

8. Modified Nucleotide Sequences Encoding Antibody Polypeptides

In some embodiments, the polynucleotide (e.g., a RNA, e.g., an mRNA) ofthe present disclosure comprises a chemically modified nucleobase, forexample, a chemically modified uracil, e.g., pseudouracil,N1-methylpseuodouracil, 5-methoxyuracil, or the like. In someembodiments, the mRNA is a uracil-modified sequence comprising an ORFencoding an antibody, wherein the mRNA comprises a chemically modifiednucleobase, for example, a chemically modified uracil, e.g.,pseudouracil, 1-methylpseuodouracil, or 5-methoxyuracil.

In certain aspects of the present disclosure, when the modified uracilbase is connected to a ribose sugar, as it is in polynucleotides, theresulting modified nucleoside or nucleotide is referred to as modifieduradine. In some embodiments, uracil in the polynucleotide is at leastabout 25%, at least about 30%, at least about 40%, at least about 50%,at least about 60%, at least about 70%, at least about 80%, at least90%, at least 95%, at least 99%, or about 100% modified uracil. In oneembodiment, uracil in the polynucleotide is at least 95% modifieduracil. In another embodiment, uracil in the polynucleotide is 100%modified uracil.

In embodiments where uracil in the polynucleotide is at least 95%modified uracil overall uracil content can be adjusted such that an mRNAprovides suitable protein expression levels while inducing little to noimmune response. In some embodiments, the uracil content of the ORF isbetween about 100% and about 150%, between about 100% and about 110%,between about 105% and about 115%, between about 110% and about 120%,between about 115% and about 125%, between about 120% and about 130%,between about 125% and about 135%, between about 130% and about 140%,between about 135% and about 145%, between about 140% and about 150% ofthe theoretical minimum uracil content in the corresponding wild-typeORF (% UTM). In other embodiments, the uracil content of the ORF isbetween about 121% and about 136% or between 123% and 134% of the % UTM.In some embodiments, the uracil content of the ORF encoding ananti-CHIKV antibody polypeptide is about 115%, about 120%, about 125%,about 130%, about 135%, about 140%, about 145%, or about 150% of the %UTM. In this context, the term “uracil” can refer to modified uraciland/or naturally occurring uracil.

In some embodiments, the uracil content in the ORF of the mRNA encodingan anti-CHIKV antibody polypeptide, as described herein, is less thanabout 30%, about 25%, about 20%, about 15%, or about 10% of the totalnucleobase content in the ORF. In some embodiments, the uracil contentin the ORF is between about 10% and about 20% of the total nucleobasecontent in the ORF. In other embodiments, the uracil content in the ORFis between about 10% and about 25% of the total nucleobase content inthe ORF. In one embodiment, the uracil content in the ORF of the mRNAencoding an anti-CHIKV antibody polypeptide is less than about 20% ofthe total nucleobase content in the open reading frame. In this context,the term “uracil” can refer to modified uracil and/or naturallyoccurring uracil.

In further embodiments, the ORF of the mRNA encoding an anti-CHIKVantibody polypeptide having modified uracil and adjusted uracil contenthas increased Cytosine (C), Guanine (G), or Guanine/Cytosine (G/C)content (absolute or relative). In some embodiments, the overallincrease in C, G, or G/C content (absolute or relative) of the ORF is atleast about 2%, at least about 3%, at least about 4%, at least about 5%,at least about 6%, at least about 7%, at least about 10%, at least about15%, at least about 20%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, or at least about 100% relative to the G/Ccontent (absolute or relative) of the wild-type ORF. In someembodiments, the G, the C, or the G/C content in the ORF is less thanabout 100%, less than about 90%, less than about 85%, or less than about80% of the theoretical maximum G, C, or G/C content of the correspondingwild type nucleotide sequence encoding the anti-CHIKV antibodypolypeptide (% GTMX; % CTMX, or % G/CTMX). In some embodiments, theincreases in G and/or C content (absolute or relative) described hereincan be conducted by replacing synonymous codons with low G, C, or G/Ccontent with synonymous codons having higher G, C, or G/C content. Inother embodiments, the increase in G and/or C content (absolute orrelative) is conducted by replacing a codon ending with U with asynonymous codon ending with G or C.

In further embodiments, the ORF of the mRNA encoding an anti-CHIKVantibody polypeptide of the invention comprises modified uracil and hasan adjusted uracil content containing less uracil pairs (UU) and/oruracil triplets (UUU) and/or uracil quadruplets (UUUU) than thecorresponding wild-type nucleotide sequence encoding the anti-CHIKVantibody polypeptide. In some embodiments, the ORF of the mRNA encodinga CHIKV antibody polypeptide, as disclosed herein, contains no uracilpairs and/or uracil triplets and/or uracil quadruplets. In someembodiments, uracil pairs and/or uracil triplets and/or uracilquadruplets are reduced below a certain threshold, e.g., no more than 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20occurrences in the ORF of the mRNA encoding the anti-CHIKV antibodypolypeptide. In a particular embodiment, the ORF of the mRNA encodingthe anti-CHIKV antibody polypeptide of the invention contains less than20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1non-phenylalanine uracil pairs and/or triplets. In another embodiment,the ORF of the mRNA encoding the anti-CHIKV antibody polypeptidecontains no non-phenylalanine uracil pairs and/or triplets.

In further embodiments, the ORF of the mRNA encoding an anti-CHIKVantibody polypeptide of the invention comprises modified uracil and hasan adjusted uracil content containing less uracil-rich clusters than acorresponding wild-type nucleotide sequence encoding the anti-CHIKVantibody polypeptide. In some embodiments, the ORF of the mRNA encodingthe anti-CHIKV antibody polypeptide of the invention containsuracil-rich clusters that are shorter in length than correspondinguracil-rich clusters in a corresponding wild-type nucleotide sequenceencoding the anti-CHIKV antibody polypeptide.

In further embodiments, alternative lower frequency codons are employed.At least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 45%, at least about 50%, at leastabout 55%, at least about 60%, at least about 65%, at least about 70%,at least about 75%, at least about 80%, at least about 85%, at leastabout 90%, at least about 95%, at least about 99%, or 100% of the codonsin the anti-CHIKV antibody polypeptide-encoding ORF of the modifieduracil-comprising mRNA are substituted with alternative codons, eachalternative codon having a codon frequency lower than the codonfrequency of the substituted codon in the synonymous codon set. The ORFalso has adjusted uracil content, as described above. In someembodiments, at least one codon in the ORF of the mRNA encoding theanti-CHIKV antibody polypeptide is substituted with an alternative codonhaving a codon frequency lower than the codon frequency of thesubstituted codon in the synonymous codon set.

In some embodiments, the adjusted uracil content, anti-CHIKV antibodypolypeptide-encoding ORF of the modified uracil-comprising mRNA exhibitsexpression levels of anti-CHIKV antibody polypeptide when administeredto a mammalian cell that are higher than expression levels of anti-CHIKVantibody polypeptide from a corresponding wild-type mRNA. In someembodiments, the mammalian cell is a mouse cell, a rat cell, or a rabbitcell. In other embodiments, the mammalian cell is a monkey cell or ahuman cell. In some embodiments, the human cell is a HeLa cell, a BJfibroblast cell, or a peripheral blood mononuclear cell (PBMC). In someembodiments, anti-CHIKV antibody polypeptide is expressed at a levelhigher than expression levels of anti-CHIKV antibody polypeptide from acorresponding wild-type mRNA when the mRNA is administered to amammalian cell in vivo. In some embodiments, the mRNA is administered tomice, rabbits, rats, monkeys, or humans. In one embodiment, mice arenull mice. In some embodiments, the mRNA is administered to mice in anamount of about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.2mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 1 mg/kg,or about 5 mg/kg. In some embodiments, the mRNA is administeredintravenously or intramuscularly. In other embodiments, the anti-CHIKVantibody polypeptide is expressed when the mRNA is administered to amammalian cell in vitro. In some embodiments, the expression isincreased by at least about 2-fold, at least about 5-fold, at leastabout 10-fold, at least about 50-fold, at least about 500-fold, at leastabout 1500-fold, or at least about 3000-fold. In other embodiments, theexpression is increased by at least about 10%, about 20%, about 30%,about 40%, about 50%, 60%, about 70%, about 80%, about 90%, or about100%.

In some embodiments, adjusted uracil content, anti-CHIKV antibodypolypeptide-encoding ORF of the modified uracil-comprising mRNA exhibitsincreased stability. In some embodiments, the mRNA exhibits increasedstability in a cell relative to the stability of a correspondingwild-type mRNA under the same conditions. In some embodiments, the mRNAexhibits increased stability including resistance to nucleases, thermalstability, and/or increased stabilization of secondary structure. Insome embodiments, increased stability exhibited by the mRNA is measuredby determining the half-life of the mRNA (e.g., in a plasma, serum,cell, or tissue sample) and/or determining the area under the curve(AUC) of the protein expression by the mRNA over time (e.g., in vitro orin vivo). An mRNA is identified as having increased stability if thehalf-life and/or the AUC is greater than the half-life and/or the AUC ofa corresponding wild-type mRNA under the same conditions.

In some embodiments, the mRNA of the present invention induces adetectably lower immune response (e.g., innate or acquired) relative tothe immune response induced by a corresponding wild-type mRNA under thesame conditions. In other embodiments, the mRNA of the presentdisclosure induces a detectably lower immune response (e.g., innate oracquired) relative to the immune response induced by an mRNA thatencodes for an anti-CHIKV antibody polypeptide but does not comprisemodified uracil under the same conditions, or relative to the immuneresponse induced by an mRNA that encodes for an anti-CHIKV antibodypolypeptide and that comprises modified uracil but that does not haveadjusted uracil content under the same conditions. The innate immuneresponse can be manifested by increased expression of pro-inflammatorycytokines, activation of intracellular PRRs (RIG-I, MDA5, etc.), celldeath, and/or termination or reduction in protein translation. In someembodiments, a reduction in the innate immune response can be measuredby expression or activity level of Type 1 interferons (e.g., IFN-α,IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and IFN-ζ) or the expressionof interferon-regulated genes such as the toll-like receptors (e.g.,TLR7 and TLR8), and/or by decreased cell death following one or moreadministrations of the mRNA of the invention into a cell.

In some embodiments, the expression of Type-1 interferons by a mammaliancell in response to the mRNA of the present disclosure is reduced by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, orgreater than 99.9% relative to a corresponding wild-type mRNA, to anmRNA that encodes an anti-CHIKV antibody polypeptide but does notcomprise modified uracil, or to an mRNA that encodes an anti-CHIKVantibody polypeptide and that comprises modified uracil but that doesnot have adjusted uracil content. In some embodiments, the interferon isIFN-β. In some embodiments, cell death frequency caused byadministration of mRNA of the present disclosure to a mammalian cell is10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell deathfrequency observed with a corresponding wild-type mRNA, an mRNA thatencodes for an anti-CHIKV antibody polypeptide but does not comprisemodified uracil, or an mRNA that encodes for an anti-CHIKV antibodypolypeptide and that comprises modified uracil but that does not haveadjusted uracil content. In some embodiments, the mammalian cell is a BJfibroblast cell. In other embodiments, the mammalian cell is asplenocyte. In some embodiments, the mammalian cell is that of a mouseor a rat. In other embodiments, the mammalian cell is that of a human.In one embodiment, the mRNA of the present disclosure does notsubstantially induce an innate immune response of a mammalian cell intowhich the mRNA is introduced.

9. Methods of Modifying Polynucleotides

The disclosure includes modified polynucleotides comprising apolynucleotide described herein (e.g., a polynucleotide, e.g. mRNA,comprising a nucleotide sequence encoding an anti-CHIKV antibodypolypeptide). The modified polynucleotides can be chemically modifiedand/or structurally modified. When the polynucleotides of the presentinvention are chemically and/or structurally modified thepolynucleotides can be referred to as “modified polynucleotides.”

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides) encoding an anti-CHIKV antibody polypeptide. A“nucleoside” refers to a compound containing a sugar molecule (e.g., apentose or ribose) or a derivative thereof in combination with anorganic base (e.g., a purine or pyrimidine) or a derivative thereof(also referred to herein as “nucleobase”). A “nucleotide” refers to anucleoside including a phosphate group. Modified nucleotides can besynthesized by any useful method, such as, for example, chemically,enzymatically, or recombinantly, to include one or more modified ornon-natural nucleosides. Polynucleotides can comprise a region orregions of linked nucleosides. Such regions can have variable backbonelinkages. The linkages can be standard phosphodiester linkages, in whichcase the polynucleotides would comprise regions of nucleotides.

The modified polynucleotides disclosed herein can comprise variousdistinct modifications. In some embodiments, the modifiedpolynucleotides contain one, two, or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedpolynucleotide, introduced to a cell can exhibit one or more desirableproperties, e.g., improved protein expression, reduced immunogenicity,or reduced degradation in the cell, as compared to an unmodifiedpolynucleotide.

In some embodiments, a polynucleotide of the present invention (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) is structurally modified. As used herein, a“structural” modification is one in which two or more linked nucleosidesare inserted, deleted, duplicated, inverted or randomized in apolynucleotide without significant chemical modification to thenucleotides themselves. Because chemical bonds will necessarily bebroken and reformed to effect a structural modification, structuralmodifications are of a chemical nature and hence are chemicalmodifications. However, structural modifications will result in adifferent sequence of nucleotides. For example, the polynucleotide“ATCG” can be chemically modified to “AT-5meC-G”. The samepolynucleotide can be structurally modified from “ATCG” to “ATCCCG”.Here, the dinucleotide “CC” has been inserted, resulting in a structuralmodification to the polynucleotide.

Therapeutic compositions of the present disclosure comprise, in someembodiments, at least one nucleic acid (e.g., RNA) having an openreading frame encoding at least one anti-CHIKV antibody polypeptide,wherein the nucleic acid comprises nucleotides and/or nucleosides thatcan be standard (unmodified) or modified as is known in the art. In someembodiments, nucleotides and nucleosides of the present disclosurecomprise modified nucleotides or nucleosides. Such modified nucleotidesand nucleosides can be naturally-occurring modified nucleotides andnucleosides or non-naturally occurring modified nucleotides andnucleosides. Such modifications can include those at the sugar,backbone, or nucleobase portion of the nucleotide and/or nucleoside asare recognized in the art.

In some embodiments, a naturally-occurring modified nucleotide ornucleotide of the disclosure is one as is generally known or recognizedin the art. Non-limiting examples of such naturally occurring modifiednucleotides and nucleotides can be found, inter alia, in the widelyrecognized MODOMICS database.

In some embodiments, a non-naturally occurring modified nucleotide ornucleoside of the disclosure is one as is generally known or recognizedin the art. Non-limiting examples of such non-naturally occurringmodified nucleotides and nucleosides can be found, inter alia, inpublished US application Nos. PCT/US2012/058519; PCT/US2013/075177;PCT/US2014/058897; PCT/US2014/058891; PCT/US2014/070413;PCT/US2015/36773; PCT/US2015/36759; PCT/US2015/36771; orPCT/IB2017/051367 all of which are incorporated by reference herein.

In some embodiments, at least one RNA (e.g., mRNA) of the presentdisclosure is not chemically modified and comprises the standardribonucleotides consisting of adenosine, guanosine, cytosine anduridine. In some embodiments, nucleotides and nucleosides of the presentdisclosure comprise standard nucleoside residues such as those presentin transcribed RNA (e.g. A, G, C, or U). In some embodiments,nucleotides and nucleosides of the present disclosure comprise standarddeoxyribonucleosides such as those present in DNA (e.g. dA, dG, dC, ordT).

Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNAnucleic acids, such as mRNA nucleic acids) can comprise standardnucleotides and nucleosides, naturally-occurring nucleotides andnucleosides, non-naturally-occurring nucleotides and nucleosides, or anycombination thereof.

Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleicacids, such as mRNA nucleic acids), in some embodiments, comprisevarious (more than one) different types of standard and/or modifiednucleotides and nucleosides. In some embodiments, a particular region ofa nucleic acid contains one, two or more (optionally different) types ofstandard and/or modified nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNAnucleic acid), introduced to a cell or organism, exhibits reduceddegradation in the cell or organism, respectively, relative to anunmodified nucleic acid comprising standard nucleotides and nucleosides.

In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNAnucleic acid), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response) relative to an unmodified nucleic acid comprisingstandard nucleotides and nucleosides.

Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), insome embodiments, comprise non-natural modified nucleotides that areintroduced during synthesis or post-synthesis of the nucleic acids toachieve desired functions or properties. The modifications may bepresent on internucleotide linkages, purine or pyrimidine bases, orsugars. The modification may be introduced with chemical synthesis orwith a polymerase enzyme at the terminal of a chain or anywhere else inthe chain. Any of the regions of a nucleic acid may be chemicallymodified.

The present disclosure provides for modified nucleosides and nucleotidesof a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids).A “nucleoside” refers to a compound containing a sugar molecule (e.g., apentose or ribose) or a derivative thereof in combination with anorganic base (e.g., a purine or pyrimidine) or a derivative thereof(also referred to herein as “nucleobase”). A “nucleotide” refers to anucleoside, including a phosphate group. Modified nucleotides may bysynthesized by any useful method, such as, for example, chemically,enzymatically, or recombinantly, to include one or more modified ornon-natural nucleosides. Nucleic acids can comprise a region or regionsof linked nucleosides. Such regions may have variable backbone linkages.The linkages can be standard phosphodiester linkages, in which case thenucleic acids would comprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures, such as, forexample, in those nucleic acids having at least one chemicalmodification. One example of such non-standard base pairing is the basepairing between the modified nucleotide inosine and adenine, cytosine oruracil. Any combination of base/sugar or linker may be incorporated intonucleic acids of the present disclosure.

In some embodiments, modified nucleobases in nucleic acids (e.g., RNAnucleic acids, such as mRNA nucleic acids) compriseN1-methylpseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ),5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and/or pseudouridine(ψ). In some embodiments, modified nucleobases in nucleic acids (e.g.,RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyluridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methylcytidine, and/or 5-methoxy cytidine. In some embodiments, thepolyribonucleotide includes a combination of at least two (e.g., 2, 3, 4or more) of any of the aforementioned modified nucleobases, includingbut not limited to chemical modifications.

In some embodiments, a RNA nucleic acid of the disclosure comprisesN1-methyl-pseudouridine (m1ψ) substitutions at one or more or alluridine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprisesN1-methyl-pseudouridine (m1ψ) substitutions at one or more or alluridine positions of the nucleic acid and 5-methyl cytidinesubstitutions at one or more or all cytidine positions of the nucleicacid.

In some embodiments, a RNA nucleic acid of the disclosure comprisespseudouridine (ψ) substitutions at one or more or all uridine positionsof the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprisespseudouridine (ψ) substitutions at one or more or all uridine positionsof the nucleic acid and 5-methyl cytidine substitutions at one or moreor all cytidine positions of the nucleic acid.

In some embodiments, a RNA nucleic acid of the disclosure comprisesuridine at one or more or all uridine positions of the nucleic acid.

In some embodiments, nucleic acids (e.g., RNA nucleic acids, such asmRNA nucleic acids) are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a nucleic acid can be uniformly modified withN1-methyl-pseudouridine, meaning that all uridine residues in the mRNAsequence are replaced with N1-methyl-pseudouridine. Similarly, a nucleicacid can be uniformly modified for any type of nucleoside residuepresent in the sequence by replacement with a modified residue such asthose set forth above.

The nucleic acids of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in anucleic acid of the disclosure, or in a predetermined sequence regionthereof (e.g., in the mRNA including or excluding the polyA tail). Insome embodiments, all nucleotides X in a nucleic acid of the presentdisclosure (or in a sequence region thereof) are modified nucleotides,wherein X may be any one of nucleotides A, G, U, C, or any one of thecombinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

The nucleic acid may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). It will be understood that anyremaining percentage is accounted for by the presence of unmodified A,G, U, or C.

The nucleic acids may contain at a minimum 1% and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10% modified nucleotides, at least 25%modified nucleotides, at least 50% modified nucleotides, at least 80%modified nucleotides, or at least 90% modified nucleotides. For example,the nucleic acids may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the nucleic acid is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). In some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe nucleic acid is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

10. Untranslated Regions (UTRs)

Translation of a polynucleotide comprising an open reading frameencoding a polypeptide can be controlled and regulated by a variety ofmechanisms that are provided by various cis-acting nucleic acidstructures. For example, naturally-occurring, cis-acting RNA elementsthat form hairpins or other higher-order (e.g., pseudoknot)intramolecular mRNA secondary structures can provide a translationalregulatory activity to a polynucleotide, wherein the RNA elementinfluences or modulates the initiation of polynucleotide translation,particularly when the RNA element is positioned in the 5′ UTR close tothe 5′-cap structure (Pelletier and Sonenberg (1985) Cell 40(3):515-526;Kozak (1986) Proc Natl Acad Sci 83:2850-2854).

Untranslated regions (UTRs) are nucleic acid sections of apolynucleotide before a start codon (5′ UTR) and after a stop codon (3′UTR) that are not translated. In some embodiments, a polynucleotide(e.g., a ribonucleic acid (RNA), e.g., a messenger RNA (mRNA)) of theinvention comprising an open reading frame (ORF) encoding an antibodyfurther comprises UTR (e.g., a 5′UTR or functional fragment thereof, a3′UTR or functional fragment thereof, or a combination thereof).

Cis-acting RNA elements can also affect translation elongation, beinginvolved in numerous frameshifting events (Namy et al., (2004) Mol Cell13(2):157-168). Internal ribosome entry sequences (IRES) representanother type of cis-acting RNA element that are typically located in 5′UTRs, but have also been reported to be found within the coding regionof naturally-occurring mRNAs (Holcik et al. (2000) Trends Genet16(10):469-473). In cellular mRNAs, IRES often coexist with the 5′-capstructure and provide mRNAs with the functional capacity to betranslated under conditions in which cap-dependent translation iscompromised (Gebauer et al., (2012) Cold Spring Harb Perspect Biol4(7):a012245). Another type of naturally-occurring cis-acting RNAelement comprises upstream open reading frames (uORFs).Naturally-occurring uORFs occur singularly or multiply within the 5′UTRs of numerous mRNAs and influence the translation of the downstreammajor ORF, usually negatively (with the notable exception of GCN4 mRNAin yeast and ATF4 mRNA in mammals, where uORFs serve to promote thetranslation of the downstream major ORF under conditions of increasedeIF2 phosphorylation (Hinnebusch (2005) Annu Rev Microbiol 59:407-450)).Additional exemplary translational regulatory activities provided bycomponents, structures, elements, motifs, and/or specific sequencescomprising polynucleotides (e.g., mRNA) include, but are not limited to,mRNA stabilization or destabilization (Baker & Parker (2004) Curr OpinCell Biol 16(3):293-299), translational activation (Villalba et al.,(2011) Curr Opin Genet Dev 21(4):452-457), and translational repression(Blumer et al., (2002) Mech Dev 110(1-2):97-112). Studies have shownthat naturally-occurring, cis-acting RNA elements can confer theirrespective functions when used to modify, by incorporation into,heterologous polynucleotides (Goldberg-Cohen et al., (2002) J Biol Chem277(16):13635-13640).

Modified Polynucleotides Comprising Functional RNA Elements

The present disclosure provides synthetic polynucleotides comprising amodification (e.g., an RNA element), wherein the modification provides adesired translational regulatory activity. In some embodiments, thedisclosure provides a polynucleotide comprising a 5′ untranslated region(UTR), an initiation codon, a full open reading frame encoding apolypeptide, a 3′ UTR, and at least one modification, wherein the atleast one modification provides a desired translational regulatoryactivity, for example, a modification that promotes and/or enhances thetranslational fidelity of mRNA translation. In some embodiments, thedesired translational regulatory activity is a cis-acting regulatoryactivity. In some embodiments, the desired translational regulatoryactivity is an increase in the residence time of the 43S pre-initiationcomplex (PIC) or ribosome at, or proximal to, the initiation codon. Insome embodiments, the desired translational regulatory activity is anincrease in the initiation of polypeptide synthesis at or from theinitiation codon. In some embodiments, the desired translationalregulatory activity is an increase in the amount of polypeptidetranslated from the full open reading frame. In some embodiments, thedesired translational regulatory activity is an increase in the fidelityof initiation codon decoding by the PIC or ribosome. In someembodiments, the desired translational regulatory activity is inhibitionor reduction of leaky scanning by the PIC or ribosome. In someembodiments, the desired translational regulatory activity is a decreasein the rate of decoding the initiation codon by the PIC or ribosome. Insome embodiments, the desired translational regulatory activity isinhibition or reduction in the initiation of polypeptide synthesis atany codon within the mRNA other than the initiation codon. In someembodiments, the desired translational regulatory activity is inhibitionor reduction of the amount of polypeptide translated from any openreading frame within the mRNA other than the full open reading frame. Insome embodiments, the desired translational regulatory activity isinhibition or reduction in the production of aberrant translationproducts. In some embodiments, the desired translational regulatoryactivity is a combination of one or more of the foregoing translationalregulatory activities.

Accordingly, the present disclosure provides a polynucleotide, e.g., anmRNA, comprising an RNA element that comprises a sequence and/or an RNAsecondary structure(s) that provides a desired translational regulatoryactivity as described herein. In some aspects, the mRNA comprises an RNAelement that comprises a sequence and/or an RNA secondary structure(s)that promotes and/or enhances the translational fidelity of mRNAtranslation. In some aspects, the mRNA comprises an RNA element thatcomprises a sequence and/or an RNA secondary structure(s) that providesa desired translational regulatory activity, such as inhibiting and/orreducing leaky scanning. In some aspects, the disclosure provides anmRNA that comprises an RNA element that comprises a sequence and/or anRNA secondary structure(s) that inhibits and/or reduces leaky scanningthereby promoting the translational fidelity of the mRNA.

In some embodiments, the RNA element comprises natural and/or modifiednucleotides. In some embodiments, the RNA element comprises of asequence of linked nucleotides, or derivatives or analogs thereof, thatprovides a desired translational regulatory activity as describedherein. In some embodiments, the RNA element comprises a sequence oflinked nucleotides, or derivatives or analogs thereof, that forms orfolds into a stable RNA secondary structure, wherein the RNA secondarystructure provides a desired translational regulatory activity asdescribed herein. RNA elements can be identified and/or characterizedbased on the primary sequence of the element (e.g., GC-rich element), byRNA secondary structure formed by the element (e.g. stem-loop), by thelocation of the element within the RNA molecule (e.g., located withinthe 5′ UTR of an mRNA), by the biological function and/or activity ofthe element (e.g., “translational enhancer element”), and anycombination thereof.

In some aspects, the disclosure provides an mRNA having one or morestructural modifications that inhibits leaky scanning and/or promotesthe translational fidelity of mRNA translation, wherein at least one ofthe structural modifications is a GC-rich RNA element. In some aspects,the disclosure provides a modified mRNA comprising at least onemodification, wherein at least one modification is a GC-rich RNA elementcomprising a sequence of linked nucleotides, or derivatives or analogsthereof, preceding a Kozak consensus sequence in a 5′ UTR of the mRNA.In one embodiment, the GC-rich RNA element is located about 30, about25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, orabout 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5′UTR of the mRNA. In another embodiment, the GC-rich RNA element islocated 15-30, 15-20, 15-25, 10-15, or 5-10 nucleotides upstream of aKozak consensus sequence. In another embodiment, the GC-rich RNA elementis located immediately adjacent to a Kozak consensus sequence in the 5′UTR of the mRNA.

In any of the foregoing or related aspects, the disclosure provides aGC-rich RNA element which comprises a sequence of 3-30, 5-25, 10-20,15-20, about 20, about 15, about 12, about 10, about 7, about 6 or about3 nucleotides, derivatives or analogs thereof, linked in any order,wherein the sequence composition is 70-80% cytosine, 60-70% cytosine,50%-60% cytosine, 40-50% cytosine, 30-40% cytosine bases. In any of theforegoing or related aspects, the disclosure provides a GC-rich RNAelement which comprises a sequence of 3-30, 5-25, 10-20, 15-20, about20, about 15, about 12, about 10, about 7, about 6 or about 3nucleotides, derivatives or analogs thereof, linked in any order,wherein the sequence composition is about 80% cytosine, about 70%cytosine, about 60% cytosine, about 50% cytosine, about 40% cytosine, orabout 30% cytosine.

In any of the foregoing or related aspects, the disclosure provides aGC-rich RNA element which comprises a sequence of 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 nucleotides, orderivatives or analogs thereof, linked in any order, wherein thesequence composition is 70-80% cytosine, 60-70% cytosine, 50%-60%cytosine, 40-50% cytosine, or 30-40% cytosine. In any of the foregoingor related aspects, the disclosure provides a GC-rich RNA element whichcomprises a sequence of 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9,8, 7, 6, 5, 4, or 3 nucleotides, or derivatives or analogs thereof,linked in any order, wherein the sequence composition is about 80%cytosine, about 70% cytosine, about 60% cytosine, about 50% cytosine,about 40% cytosine, or about 30% cytosine.

In some embodiments, the disclosure provides a modified mRNA comprisingat least one modification, wherein at least one modification is aGC-rich RNA element comprising a sequence of linked nucleotides, orderivatives or analogs thereof, preceding a Kozak consensus sequence ina 5′ UTR of the mRNA, wherein the GC-rich RNA element is located about30, about 25, about 20, about 15, about 10, about 5, about 4, about 3,about 2, or about 1 nucleotide(s) upstream of a Kozak consensus sequencein the 5′ UTR of the mRNA, and wherein the GC-rich RNA element comprisesa sequence of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, or 20 nucleotides, or derivatives or analogs thereof, linked in anyorder, wherein the sequence composition is >50% cytosine. In someembodiments, the sequence composition is >55% cytosine, >60%cytosine, >65% cytosine, >70% cytosine, >75% cytosine, >80%cytosine, >85% cytosine, or >90% cytosine.

In other aspects, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising a sequence of linked nucleotides, or derivativesor analogs thereof, preceding a Kozak consensus sequence in a 5′ UTR ofthe mRNA, wherein the GC-rich RNA element is located about 30, about 25,about 20, about 15, about 10, about 5, about 4, about 3, about 2, orabout 1 nucleotide(s) upstream of a Kozak consensus sequence in the 5′UTR of the mRNA, and wherein the GC-rich RNA element comprises asequence of about 3-30, 5-25, 10-20, 15-20 or about 20, about 15, about12, about 10, about 6 or about 3 nucleotides, or derivatives oranalogues thereof, wherein the sequence comprises a repeating GC-motif,wherein the repeating GC-motif is [CCG]n, wherein n=1 to 10, n=2 to 8,n=3 to 6, or n=4 to 5. In some embodiments, the sequence comprises arepeating GC-motif [CCG]n, wherein n=1, 2, 3, 4 or 5. In someembodiments, the sequence comprises a repeating GC-motif [CCG]n, whereinn=1, 2, or 3. In some embodiments, the sequence comprises a repeatingGC-motif [CCG]n, wherein n=1. In some embodiments, the sequencecomprises a repeating GC-motif [CCG]n, wherein n=2. In some embodiments,the sequence comprises a repeating GC-motif [CCG]n, wherein n=3. In someembodiments, the sequence comprises a repeating GC-motif [CCG]n, whereinn=4. In some embodiments, the sequence comprises a repeating GC-motif[CCG]n, wherein n=5.

In another aspect, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising a sequence of linked nucleotides, or derivativesor analogs thereof, preceding a Kozak consensus sequence in a 5′ UTR ofthe mRNA, wherein the GC-rich RNA element comprises any one of thesequences set forth in Table 2. In one embodiment, the GC-rich RNAelement is located about 30, about 25, about 20, about 15, about 10,about 5, about 4, about 3, about 2, or about 1 nucleotide(s) upstream ofa Kozak consensus sequence in the 5′ UTR of the mRNA. In anotherembodiment, the GC-rich RNA element is located about 15-30, 15-20,15-25, 10-15, or 5-10 nucleotides upstream of a Kozak consensussequence. In another embodiment, the GC-rich RNA element is locatedimmediately adjacent to a Kozak consensus sequence in the 5′ UTR of themRNA.

In other aspects, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising the sequence V1 [CCCCGGCGCC (SEQ ID NO: 100)] asset forth in Table 2, or derivatives or analogs thereof, preceding aKozak consensus sequence in the 5′ UTR of the mRNA. In some embodiments,the GC-rich element comprises the sequence V1 as set forth in Table 2located immediately adjacent to and upstream of the Kozak consensussequence in the 5′ UTR of the mRNA. In some embodiments, the GC-richelement comprises the sequence V1 as set forth in Table 2 located 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequencein the 5′ UTR of the mRNA. In other embodiments, the GC-rich elementcomprises the sequence V1 as set forth in Table 2 located 1-3, 3-5, 5-7,7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence inthe 5′ UTR of the mRNA. In other aspects, the disclosure provides amodified mRNA comprising at least one modification, wherein at least onemodification is a GC-rich RNA element comprising the sequence V2[CCCCGGC (SEQ ID NO: 101)] as set forth in Table 2, or derivatives oranalogs thereof, preceding a Kozak consensus sequence in the 5′ UTR ofthe mRNA. In some embodiments, the GC-rich element comprises thesequence V2 as set forth in Table 2 located immediately adjacent to andupstream of the Kozak consensus sequence in the 5′ UTR of the mRNA. Insome embodiments, the GC-rich element comprises the sequence V2 as setforth in Table 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstreamof the Kozak consensus sequence in the 5′ UTR of the mRNA. In otherembodiments, the GC-rich element comprises the sequence V2 as set forthin Table 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 bases upstream ofthe Kozak consensus sequence in the 5′ UTR of the mRNA.

In other aspects, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising the sequence EK [GCCGCC (SEQ ID NO: 102)] as setforth in Table 2, or derivatives or analogs thereof, preceding a Kozakconsensus sequence in the 5′ UTR of the mRNA. In some embodiments, theGC-rich element comprises the sequence EK as set forth in Table 2located immediately adjacent to and upstream of the Kozak consensussequence in the 5′ UTR of the mRNA. In some embodiments, the GC-richelement comprises the sequence EK as set forth in Table 2 located 1, 2,3, 4, 5, 6, 7, 8, 9 or 10 bases upstream of the Kozak consensus sequencein the 5′ UTR of the mRNA. In other embodiments, the GC-rich elementcomprises the sequence EK as set forth in Table 2 located 1-3, 3-5, 5-7,7-9, 9-12, or 12-15 bases upstream of the Kozak consensus sequence inthe 5′ UTR of the mRNA.

In yet other aspects, the disclosure provides a modified mRNA comprisingat least one modification, wherein at least one modification is aGC-rich RNA element comprising the sequence V1 [CCCCGGCGCC (SEQ ID NO:100)] as set forth in Table 2, or derivatives or analogs thereof,preceding a Kozak consensus sequence in the 5′ UTR of the mRNA, whereinthe 5′ UTR comprises the following sequence shown in Table 2:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO: 103). The skilledartisan will of course recognize that all Us in the RNA sequencesdescribed herein will be Ts in a corresponding template DNA sequence,for example, in DNA templates or constructs from which mRNAs of thedisclosure are transcribed, e.g., via IVT.

In some embodiments, the GC-rich element comprises the sequence V1 asset forth in Table 2 located immediately adjacent to and upstream of theKozak consensus sequence in the 5′ UTR sequence shown in Table 2. Insome embodiments, the GC-rich element comprises the sequence V1 as setforth in Table 2 located 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases upstreamof the Kozak consensus sequence in the 5′ UTR of the mRNA, wherein the5′ UTR comprises the following sequence shown in Table 2:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO: 103).

In other embodiments, the GC-rich element comprises the sequence V1 asset forth in Table 2 located 1-3, 3-5, 5-7, 7-9, 9-12, or 12-15 basesupstream of the Kozak consensus sequence in the 5′ UTR of the mRNA,wherein the 5′ UTR comprises the following sequence shown in Table 2:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA (SEQ ID NO: 103).

In some embodiments, the 5′ UTR comprises the following sequence setforth in Table 2:

(SEQ ID NO: 13) GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCCACC

TABLE 2 5′ UTRs 5′ UTR Sequence Standard GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 108) V1-UTR GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGC CGCCACC (SEQ ID NO: 13) V2-UTRGGGAAAUAAGAGAGAAAAGAAGAGUAA GAAGAAAUAUAAGACCCCGGCGCCACC (SEQ ID NO: 106)GC-Rich RNA Elements Sequence K0 (Traditional[GCCA/GCC] (SEQ ID NO: 107) Kozak consensus) EK[GCCGCC] (SEQ ID NO: 102) V1 [CCCCGGCGCC] (SEQ ID NO: 100) V2[CCCCGGC] (SEQ ID NO: 101) (CCG)_(n), where n = 1-10 [CCG]_(n)(GCC)_(n), where n = 1-10 [GCC]_(n)

In another aspect, the disclosure provides a modified mRNA comprising atleast one modification, wherein at least one modification is a GC-richRNA element comprising a stable RNA secondary structure comprising asequence of nucleotides, or derivatives or analogs thereof, linked in anorder which forms a hairpin or a stem-loop. In one embodiment, thestable RNA secondary structure is upstream of the Kozak consensussequence. In another embodiment, the stable RNA secondary structure islocated about 30, about 25, about 20, about 15, about 10, or about 5nucleotides upstream of the Kozak consensus sequence. In anotherembodiment, the stable RNA secondary structure is located about 20,about 15, about 10 or about 5 nucleotides upstream of the Kozakconsensus sequence. In another embodiment, the stable RNA secondarystructure is located about 5, about 4, about 3, about 2, about 1nucleotides upstream of the Kozak consensus sequence. In anotherembodiment, the stable RNA secondary structure is located about 15-30,about 15-20, about 15-25, about 10-15, or about 5-10 nucleotidesupstream of the Kozak consensus sequence. In another embodiment, thestable RNA secondary structure is located 12-15 nucleotides upstream ofthe Kozak consensus sequence. In another embodiment, the stable RNAsecondary structure has a deltaG of about −30 kcal/mol, about −20 to −30kcal/mol, about −20 kcal/mol, about −10 to −20 kcal/mol, about −10kcal/mol, about −5 to −10 kcal/mol.

In another embodiment, the modification is operably linked to an openreading frame encoding a polypeptide and wherein the modification andthe open reading frame are heterologous.

In another embodiment, the sequence of the GC-rich RNA element iscomprised exclusively of guanine (G) and cytosine (C) nucleobases.

RNA elements that provide a desired translational regulatory activity asdescribed herein can be identified and characterized using knowntechniques, such as ribosome profiling. Ribosome profiling is atechnique that allows the determination of the positions of PICs and/orribosomes bound to mRNAs (see e.g., Ingolia et al., (2009) Science324(5924):218-23, incorporated herein by reference). The technique isbased on protecting a region or segment of mRNA, by the PIC and/orribosome, from nuclease digestion. Protection results in the generationof a 30-bp fragment of RNA termed a ‘footprint’. The sequence andfrequency of RNA footprints can be analyzed by methods known in the art(e.g., RNA-seq). The footprint is roughly centered on the A-site of theribosome. If the PIC or ribosome dwells at a particular position orlocation along an mRNA, footprints generated at these position would berelatively common. Studies have shown that more footprints are generatedat positions where the PIC and/or ribosome exhibits decreasedprocessivity and fewer footprints where the PIC and/or ribosome exhibitsincreased processivity (Gardin et al., (2014) eLife 3:e03735). In someembodiments, residence time or the time of occupancy of the PIC orribosome at a discrete position or location along an polynucleotidecomprising any one or more of the RNA elements described herein isdetermined by ribosome profiling.

A UTR can be homologous or heterologous to the coding region in apolynucleotide. In some embodiments, the UTR is homologous to the ORFencoding the antibody. In some embodiments, the UTR is heterologous tothe ORF encoding the antibody. In some embodiments, the polynucleotidecomprises two or more 5′UTRs or functional fragments thereof, each ofwhich have the same or different nucleotide sequences. In someembodiments, the polynucleotide comprises two or more 3′UTRs orfunctional fragments thereof, each of which have the same or differentnucleotide sequences.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR orfunctional fragment thereof, or any combination thereof is sequenceoptimized.

In some embodiments, the 5′UTR or functional fragment thereof, 3′ UTR orfunctional fragment thereof, or any combination thereof comprises atleast one chemically modified nucleobase, e.g., N1 methylpseudouracil or5-methoxyuracil.

UTRs can have features that provide a regulatory role, e.g., increasedor decreased stability, localization and/or translation efficiency. Apolynucleotide comprising a UTR can be administered to a cell, tissue,or organism, and one or more regulatory features can be measured usingroutine methods. In some embodiments, a functional fragment of a 5′UTRor 3′UTR comprises one or more regulatory features of a full length 5′or 3′ UTR, respectively.

Natural 5′UTRs bear features that play roles in translation initiation.They harbor signatures like Kozak sequences that are commonly known tobe involved in the process by which the ribosome initiates translationof many genes. Kozak sequences have the consensus CCR(A/G)CCAUGG (SEQ IDNO: 231, where R is a purine (adenine or guanine) three bases upstreamof the start codon (AUG), which is followed by another ‘G’. 5′UTRs alsohave been known to form secondary structures that are involved inelongation factor binding.

By engineering the features typically found in abundantly expressedgenes of specific target organs, one can enhance the stability andprotein production of a polynucleotide. For example, introduction of5′UTR of liver-expressed mRNA, such as albumin, serum amyloid A,Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, orantibody, can enhance expression of polynucleotides in hepatic celllines or liver. Likewise, use of 5′UTR from other tissue-specific mRNAto improve expression in that tissue is possible for muscle (e.g., MyoD,Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (e.g.,Tie-1, CD36), for myeloid cells (e.g., C/EBP, AML1, G-CSF, GM-CSF,CD11b, MSR, Fr-1, i-NOS), for leukocytes (e.g., CD45, CD18), for adiposetissue (e.g., CD36, GLUT4, ACRP30, adiponectin) and for lung epithelialcells (e.g., SP-A/B/C/D).

In some embodiments, UTRs are selected from a family of transcriptswhose proteins share a common function, structure, feature or property.For example, an encoded polypeptide can belong to a family of proteins(i.e., that share at least one function, structure, feature,localization, origin, or expression pattern), which are expressed in aparticular cell, tissue or at some time during development. The UTRsfrom any of the genes or mRNA can be swapped for any other UTR of thesame or different family of proteins to create a new polynucleotide.

In some embodiments, the 5′UTR and the 3′UTR can be heterologous. Insome embodiments, the 5′UTR can be derived from a different species thanthe 3′UTR. In some embodiments, the 3′UTR can be derived from adifferent species than the 5′UTR.

Co-owned International Patent Application No. PCT/US2014/021522 (Publ.No. WO/2014/164253, incorporated herein by reference in its entirety)provides a listing of exemplary UTRs that can be utilized in thepolynucleotide of the present disclosure as flanking regions to an ORF.

Exemplary UTRs of the application include, but are not limited to, oneor more 5′UTR and/or 3′UTR derived from the nucleic acid sequence of: aglobin, such as an α- or β-globin (e.g., a Xenopus, mouse, rabbit, orhuman globin); a strong Kozak translational initiation signal; a CYBA(e.g., human cytochrome b-245 α polypeptide); an albumin (e.g., humanalbumin7); a HSD17B4 (hydroxysteroid (17-β) dehydrogenase); a virus(e.g., a tobacco etch virus (TEV), a Venezuelan equine encephalitisvirus (VEEV), a Dengue virus, a cytomegalovirus (CMV) (e.g., CMVimmediate early 1 (IE1)), a hepatitis virus (e.g., hepatitis B virus), asindbis virus, or a PAV barley yellow dwarf virus); a heat shock protein(e.g., hsp70); a translation initiation factor (e.g., elF4G); a glucosetransporter (e.g., hGLUT1 (human glucose transporter 1)); an actin(e.g., human α or β actin); a GAPDH; a tubulin; a histone; a citric acidcycle enzyme; a topoisomerase (e.g., a 5′UTR of a TOP gene lacking the5′ TOP motif (the oligopyrimidine tract)); a ribosomal protein Large 32(L32); a ribosomal protein (e.g., human or mouse ribosomal protein, suchas, for example, rps9); an ATP synthase (e.g., ATP5A1 or the β subunitof mitochondrial H⁺-ATP synthase); a growth hormone e (e.g., bovine(bGH) or human (hGH)); an elongation factor (e.g., elongation factor 1α1 (EEF1A1)); a manganese superoxide dismutase (MnSOD); a myocyteenhancer factor 2A (MEF2A); a β-F1-ATPase, a creatine kinase, amyoglobin, a granulocyte-colony stimulating factor (G-CSF); a collagen(e.g., collagen type I, alpha 2 (Col1A2), collagen type I, alpha 1(Col1A1), collagen type VI, alpha 2 (Col6A2), collagen type VI, alpha 1(Col6A1)); a ribophorin (e.g., ribophorin I (RPNI)); a low densitylipoprotein receptor-related protein (e.g., LRP1); a cardiotrophin-likecytokine factor (e.g., Nnt1); calreticulin (Calr); a procollagen-lysine,2-oxoglutarate 5-dioxygenase 1 (Plod1); and a nucleobindin (e.g.,Nucb1).

In some embodiments, the 5′UTR is selected from the group consisting ofa β-globin 5′UTR; a 5′UTR containing a strong Kozak translationalinitiation signal; a cytochrome b-245 α polypeptide (CYBA) 5′UTR; ahydroxysteroid (17-β) dehydrogenase (HSD17B4) 5′UTR; a Tobacco etchvirus (TEV) 5′UTR; a Venezuelen equine encephalitis virus (TEEV) 5′UTR;a 5′ proximal open reading frame of rubella virus (RV) RNA encodingnonstructural proteins; a Dengue virus (DEN) 5′UTR; a heat shock protein70 (Hsp70) 5′UTR; a eIF4G 5′UTR; a GLUT1 5′UTR; functional fragmentsthereof and any combination thereof.

In some embodiments, the 3′UTR is selected from the group consisting ofa β-globin 3′UTR; a CYBA 3′UTR; an albumin 3′UTR; a growth hormone (GH)3′UTR; a VEEV 3′UTR; a hepatitis B virus (HBV) 3′UTR; α-globin 3′UTR; aDEN 3′UTR; a PAV barley yellow dwarf virus (BYDV-PAV) 3′UTR; anelongation factor 1 α1 (EEF1A1) 3′UTR; a manganese superoxide dismutase(MnSOD) 3′UTR; a β subunit of mitochondrial H(+)-ATP synthase (β-mRNA)3′UTR; a GLUT1 3′UTR; a MEF2A 3′UTR; a β-F1-ATPase 3′UTR; functionalfragments thereof and combinations thereof.

Wild-type UTRs derived from any gene or mRNA can be incorporated intothe polynucleotides of the present disclosure. In some embodiments, aUTR can be altered relative to a wild type or native UTR to produce avariant UTR, e.g., by changing the orientation or location of the UTRrelative to the ORF; or by inclusion of additional nucleotides, deletionof nucleotides, swapping or transposition of nucleotides. In someembodiments, variants of 5′ or 3′ UTRs can be utilized, for example,mutants of wild type UTRs, or variants wherein one or more nucleotidesare added to or removed from a terminus of the UTR.

Additionally, one or more synthetic UTRs can be used in combination withone or more non-synthetic UTRs. See, e.g., Mandal and Rossi, Nat.Protoc. 2013 8(3):568-82, the contents of which are incorporated hereinby reference in their entirety.

UTRs or portions thereof can be placed in the same orientation as in thetranscript from which they were selected or can be altered inorientation or location. Hence, a 5′ and/or 3′ UTR can be inverted,shortened, lengthened, or combined with one or more other 5′ UTRs or 3′UTRs.

In some embodiments, the polynucleotide comprises multiple UTRs, e.g., adouble, a triple or a quadruple 5′UTR or 3′UTR. For example, a doubleUTR comprises two copies of the same UTR either in series orsubstantially in series. For example, a double beta-globin 3′UTR can beused (see US2010/0129877, the contents of which are incorporated hereinby reference in its entirety).

In certain embodiments, the polynucleotides of the invention comprise a5′ UTR and/or a 3′ UTR selected from any of the UTRs disclosed herein.In some embodiments, the 5′ UTR comprises:

5′ UTR-001 (Upstream UTR) (SEQ ID NO: 108)(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-002 (Upstream UTR) (SEQ ID NO: 109)(GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-003 (Upstream UTR) (SEQ ID NO: 110) (See WO2016/100812);5′ UTR-004 (Upstream UTR) (SEQ ID NO: 111)(GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC); 5′ UTR-005 (Upstream UTR)(SEQ ID NO: 109) (GGGAGAUCAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-006 (Upstream UTR) (SEQ ID NO: 113) (See WO2016/100812);5′ UTR-007 (Upstream UTR) (SEQ ID NO: 111)(GGGAGACAAGCUUGGCAUUCCGGUACUGUUGGUAAAGCCACC); 5′ UTR-008 (Upstream UTR)(SEQ ID NO: 115) (GGGAAUUAACAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-009 (Upstream UTR) (SEQ ID NO: 116)(GGGAAAUUAGACAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC); 5′ UTR-010, Upstream(SEQ ID NO: 117) (GGGAAAUAAGAGAGUAAAGAACAGUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-011 (Upstream UTR) (SEQ ID NO: 118)(GGGAAAAAAGAGAGAAAAGAAGACUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-012 (Upstream UTR) (SEQ ID NO: 119)(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAUAUAUAAGAGCCACC);5′ UTR-013 (Upstream UTR) (SEQ ID NO: 120)(GGGAAAUAAGAGACAAAACAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-014 (Upstream UTR) (SEQ ID NO: 121)(GGGAAAUUAGAGAGUAAAGAACAGUAAGUAGAAUUAAAAGAGCCACC);5′ UTR-015 (Upstream UTR) (SEQ ID NO: 122)(GGGAAAUAAGAGAGAAUAGAAGAGUAAGAAGAAAUAUAAGAGCCACC);5′ UTR-016 (Upstream UTR) (SEQ ID NO: 123)(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAAUUAAGAGCCACC);5′ UTR-017 (Upstream UTR); or (SEQ ID NO: 124)(GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUUUAAGAGCCACC);5′ UTR-018 (Upstream UTR) 5′ UTR (SEQ ID NO: 126)(UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC).

In some embodiments, the 3′ UTR comprises:

142-3p 3′ UTR (UTR including miR142-3p binding site) (SEQ ID NO: 127)(UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3′ UTR(UTR including miR142-3p binding site) (SEQ ID NO: 128)(UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); or 142-3p 3′ UTR(UTR including miR142-3p binding site) (SEQ ID NO: 129)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAAAGUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3′ UTR(UTR including miR142-3p binding site) (SEQ ID NO: 130)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3′ UTR(UTR including miR142-3p binding site) (SEQ ID NO: 131)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC); 142-3p 3′ UTR(UTR including miR142-3p binding site) (SEQ ID NO: 132)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC). 142-3p 3′ UTR(UTR including miR142-3p binding site) (SEQ ID NO: 133)(UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC);3'UTR-018 (See SEQ ID NO: 134)3′ UTR (miR142 and miR126 binding sites variant 1) (SEQ ID NO: 135)(UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGC)3′ UTR (miR142 and miR126 binding sites variant 2) (SEQ ID NO: 136)(UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCCGCAUUAUUACUCACGGUACGAGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGC); or3′ UTR (miR142-3p binding site variant 3) (SEQ ID NO: 137)UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC.3′ UTR (miR142-3p binding site variant 3. DNA sequence) (SEQ ID NO: 138)TGATAATAGGCTGGAGCCTCGGTGGCCTAGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCTCCATAAAGTAGGAAACACTACAGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC.

In certain embodiments, the 5′UTR and/or 3′UTR sequence of the presentdisclosure comprises a nucleotide sequence at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or about 100% identical to a sequence selected from the groupconsisting of 5′UTR sequences comprising any of SEQ ID NOs: 13 and108-126 and/or 3′UTR sequences comprises any of SEQ ID NOs: 14 and127-138, and any combination thereof. In certain embodiments, the 5′ UTRsequence useful for the invention comprises a nucleotide sequence atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or about 100% identical to SEQ IDNO: 13. In certain embodiments, the 3′ UTR sequence useful for theinvention comprises a nucleotide sequence at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 96%, at least about 97%, at least about 98%, at leastabout 99%, or about 100% identical to SEQ ID NO: 14.

The polynucleotides of the present disclosure can comprise combinationsof features. For example, the ORF can be flanked by a 5′UTR thatcomprises a strong Kozak translational initiation signal and/or a 3′UTRcomprising an oligo(dT) sequence for templated addition of a poly-Atail. A 5′UTR can comprise a first polynucleotide fragment and a secondpolynucleotide fragment from the same and/or different UTRs (see, e.g.,US2010/0293625, herein incorporated by reference in its entirety).

Other non-UTR sequences can be used as regions or subregions within thepolynucleotides of the present disclosure. For example, introns orportions of intron sequences can be incorporated into thepolynucleotides of the present disclosure. Incorporation of intronicsequences can increase protein production as well as polynucleotideexpression levels. In some embodiments, the polynucleotide of thepresent disclosure comprises an internal ribosome entry site (IRES)instead of or in addition to a UTR (see, e.g., Yakubov et al., Biochem.Biophys. Res. Commun. 2010 394(1):189-193, the contents of which areincorporated herein by reference in their entirety). In someembodiments, the polynucleotide comprises an IRES instead of a 5′UTRsequence. In some embodiments, the polynucleotide comprises an ORF and aviral capsid sequence. In some embodiments, the polynucleotide comprisesa synthetic 5′UTR in combination with a non-synthetic 3′UTR.

In some embodiments, the UTR can also include at least one translationenhancer polynucleotide, translation enhancer element, or translationalenhancer elements (collectively, “TEE,” which refers to nucleic acidsequences that increase the amount of polypeptide or protein producedfrom a polynucleotide. As a non-limiting example, the TEE can be locatedbetween the transcription promoter and the start codon. In someembodiments, the 5′UTR comprises a TEE.

In one aspect, a TEE is a conserved element in a UTR that can promotetranslational activity of a nucleic acid such as, but not limited to,cap-dependent or cap-independent translation.

In some embodiments, a 5′UTR and/or 3′UTR comprising at least one TEEdescribed herein can be incorporated in a monocistronic sequence suchas, but not limited to, a vector system or a nucleic acid vector.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thepresent disclosure comprises a TEE or portion thereof described herein.In some embodiments, the TEEs in the 3′UTR can be the same and/ordifferent from the TEE located in the 5′UTR.

In some embodiments, a 5′UTR and/or 3′UTR of a polynucleotide of thepresent disclosure can include at least 1, at least 2, at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, atleast 10, at least 11, at least 12, at least 13, at least 14, at least15, at least 16, at least 17, at least 18 at least 19, at least 20, atleast 21, at least 22, at least 23, at least 24, at least 25, at least30, at least 35, at least 40, at least 45, at least 50, at least 55 ormore than 60 TEE sequences. In one embodiment, the 5′UTR of apolynucleotide of the present disclosure can include 1-60, 1-55, 1-50,1-45, 1-40, 1-35, 1-30, 1-25, 1-20, 1-15, 1-10, 9, 8, 7, 6, 5, 4, 3, 2,or 1 TEE sequences. The TEE sequences in the 5′UTR of the polynucleotideof the present disclosure can be the same or different TEE sequences. Acombination of different TEE sequences in the 5′UTR of thepolynucleotide of the present disclosure can include combinations inwhich more than one copy of any of the different TEE sequences areincorporated.

In some embodiments, the 5′UTR and/or 3′UTR comprises a spacer toseparate two TEE sequences. As a non-limiting example, the spacer can bea 15 nucleotide spacer and/or other spacers known in the art. As anothernon-limiting example, the 5′UTR and/or 3′UTR comprises a TEEsequence-spacer module repeated at least once, at least twice, at least3 times, at least 4 times, at least 5 times, at least 6 times, at least7 times, at least 8 times, at least 9 times, at least 10 times, or morethan 10 times in the 5′UTR and/or 3′UTR, respectively. In someembodiments, the 5′UTR and/or 3′UTR comprises a TEE sequence-spacermodule repeated 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

In some embodiments, the spacer separating two TEE sequences can includeother sequences known in the art that can regulate the translation ofthe polynucleotide of the present disclosure, e.g., miR binding sitesequences described herein (e.g., miR binding sites and miR seeds). As anon-limiting example, each spacer used to separate two TEE sequences caninclude a different miR binding site sequence or component of a miRsequence (e.g., miR seed sequence).

In some embodiments, a polynucleotide of the present disclosurecomprises a miR and/or TEE sequence. In some embodiments, theincorporation of a miR sequence and/or a TEE sequence into apolynucleotide of the present disclosure can change the shape of thestem loop region, which can increase and/or decrease translation. Seee.g., Kedde et al., Nature Cell Biology 2010 12(10):1014-20, hereinincorporated by reference in its entirety).

11. MicroRNA (miRNA) Binding Sites

Polynucleotides of the present disclosure can include regulatoryelements, for example, microRNA (miRNA) binding sites, transcriptionfactor binding sites, structured mRNA sequences and/or motifs,artificial binding sites engineered to act as pseudo-receptors forendogenous nucleic acid binding molecules, and combinations thereof. Insome embodiments, polynucleotides including such regulatory elements arereferred to as including “sensor sequences”.

In some embodiments, a polynucleotide (e.g., a ribonucleic acid (RNA),e.g., a messenger RNA (mRNA)) of the present disclosure comprises anopen reading frame (ORF) encoding a polypeptide of interest and furthercomprises one or more miRNA binding site(s). Inclusion or incorporationof miRNA binding site(s) provides for regulation of polynucleotides ofthe present disclosure, and in turn, of the polypeptides encodedtherefrom, based on tissue-specific and/or cell-type specific expressionof naturally-occurring miRNAs.

A miRNA, e.g., a natural-occurring miRNA, is a 19-25 nucleotide longnoncoding RNA that binds to a polynucleotide and down-regulates geneexpression either by reducing stability or by inhibiting translation ofthe polynucleotide. A miRNA sequence comprises a “seed” region, i.e., asequence in the region of positions 2-8 of the mature miRNA. A miRNAseed can comprise positions 2-8 or 2-7 of the mature miRNA.

microRNAs derive enzymatically from regions of RNA transcripts that foldback on themselves to form short hairpin structures often termed apre-miRNA (precursor-miRNA). A pre-miRNA typically has a two-nucleotideoverhang at its 3′ end, and has 3′ hydroxyl and 5′ phosphate groups.This precursor-mRNA is processed in the nucleus and subsequentlytransported to the cytoplasm where it is further processed by DICER (aRNase III enzyme), to form a mature microRNA of approximately 22nucleotides. The mature microRNA is then incorporated into a ribonuclearparticle to form the RNA-induced silencing complex, RISC, which mediatesgene silencing. Art-recognized nomenclature for mature miRNAs typicallydesignates the arm of the pre-miRNA from which the mature miRNA derives;“5p” means the microRNA is from the 5 prime arm of the pre-miRNA hairpinand “3p” means the microRNA is from the 3 prime end of the pre-miRNAhairpin. A miR referred to by number herein can refer to either of thetwo mature microRNAs originating from opposite arms of the samepre-miRNA (e.g., either the 3p or 5p microRNA). All miRs referred toherein are intended to include both the 3p and 5p arms/sequences, unlessparticularly specified by the 3p or 5p designation.

As used herein, the term “microRNA (miRNA or miR) binding site” refersto a sequence within a polynucleotide, e.g., within a DNA or within anRNA transcript, including in the 5′UTR and/or 3′UTR, that has sufficientcomplementarity to all or a region of a miRNA to interact with,associate with or bind to the miRNA. In some embodiments, apolynucleotide of the present disclosure comprising an ORF encoding apolypeptide of interest and further comprises one or more miRNA bindingsite(s). In exemplary embodiments, a 5′UTR and/or 3′UTR of thepolynucleotide (e.g., a ribonucleic acid (RNA), e.g., a messenger RNA(mRNA)) comprises the one or more miRNA binding site(s).

A miRNA binding site having sufficient complementarity to a miRNA refersto a degree of complementarity sufficient to facilitate miRNA-mediatedregulation of a polynucleotide, e.g., miRNA-mediated translationalrepression or degradation of the polynucleotide. In exemplary aspects ofthe present disclosure, a miRNA binding site having sufficientcomplementarity to the miRNA refers to a degree of complementaritysufficient to facilitate miRNA-mediated degradation of thepolynucleotide, e.g., miRNA-guided RNA-induced silencing complex(RISC)-mediated cleavage of mRNA. The miRNA binding site can havecomplementarity to, for example, a 19-25 nucleotide miRNA sequence, to a19-23 nucleotide miRNA sequence, or to a 22 nucleotide miRNA sequence. AmiRNA binding site can be complementary to only a portion of a miRNA,e.g., to a portion less than 1, 2, 3, or 4 nucleotides of the fulllength of a naturally-occurring miRNA sequence. Full or completecomplementarity (e.g., full complementarity or complete complementarityover all or a significant portion of the length of a naturally-occurringmiRNA) is preferred when the desired regulation is mRNA degradation.

In some embodiments, a miRNA binding site includes a sequence that hascomplementarity (e.g., partial or complete complementarity) with anmiRNA seed sequence. In some embodiments, the miRNA binding siteincludes a sequence that has complete complementarity with a miRNA seedsequence. In some embodiments, a miRNA binding site includes a sequencethat has complementarity (e.g., partial or complete complementarity)with an miRNA sequence. In some embodiments, the miRNA binding siteincludes a sequence that has complete complementarity with a miRNAsequence. In some embodiments, a miRNA binding site has completecomplementarity with a miRNA sequence but for 1, 2, or 3 nucleotidesubstitutions, terminal additions, and/or truncations.

In some embodiments, the miRNA binding site is the same length as thecorresponding miRNA. In other embodiments, the miRNA binding site isone, two, three, four, five, six, seven, eight, nine, ten, eleven ortwelve nucleotide(s) shorter than the corresponding miRNA at the 5′terminus, the 3′ terminus, or both. In still other embodiments, themicroRNA binding site is two nucleotides shorter than the correspondingmicroRNA at the 5′ terminus, the 3′ terminus, or both. The miRNA bindingsites that are shorter than the corresponding miRNAs are still capableof degrading the mRNA incorporating one or more of the miRNA bindingsites or preventing the mRNA from translation.

In some embodiments, the miRNA binding site binds the correspondingmature miRNA that is part of an active RISC containing Dicer. In anotherembodiment, binding of the miRNA binding site to the corresponding miRNAin RISC degrades the mRNA containing the miRNA binding site or preventsthe mRNA from being translated. In some embodiments, the miRNA bindingsite has sufficient complementarity to miRNA so that a RISC complexcomprising the miRNA cleaves the polynucleotide comprising the miRNAbinding site. In other embodiments, the miRNA binding site has imperfectcomplementarity so that a RISC complex comprising the miRNA inducesinstability in the polynucleotide comprising the miRNA binding site. Inanother embodiment, the miRNA binding site has imperfect complementarityso that a RISC complex comprising the miRNA represses transcription ofthe polynucleotide comprising the miRNA binding site.

In some embodiments, the miRNA binding site has one, two, three, four,five, six, seven, eight, nine, ten, eleven or twelve mismatch(es) fromthe corresponding miRNA.

In some embodiments, the miRNA binding site has at least about ten, atleast about eleven, at least about twelve, at least about thirteen, atleast about fourteen, at least about fifteen, at least about sixteen, atleast about seventeen, at least about eighteen, at least about nineteen,at least about twenty, or at least about twenty-one contiguousnucleotides complementary to at least about ten, at least about eleven,at least about twelve, at least about thirteen, at least about fourteen,at least about fifteen, at least about sixteen, at least aboutseventeen, at least about eighteen, at least about nineteen, at leastabout twenty, or at least about twenty-one, respectively, contiguousnucleotides of the corresponding miRNA.

By engineering one or more miRNA binding sites into a polynucleotide ofthe present disclosure, the polynucleotide can be targeted fordegradation or reduced translation, provided the miRNA in question isavailable. This can reduce off-target effects upon delivery of thepolynucleotide. For example, if a polynucleotide of the presentdisclosure is not intended to be delivered to a tissue or cell but endsup is said tissue or cell, then a miRNA abundant in the tissue or cellcan inhibit the expression of the gene of interest if one or multiplebinding sites of the miRNA are engineered into the 5′UTR and/or 3′UTR ofthe polynucleotide. Thus, in some embodiments, incorporation of one ormore miRNA binding sites into an mRNA of the disclosure may reduce thehazard of off-target effects upon nucleic acid molecule delivery and/orenable tissue-specific regulation of expression of a polypeptide encodedby the mRNA. In yet other embodiments, incorporation of one or moremiRNA binding sites into an mRNA of the disclosure can modulate immuneresponses upon nucleic acid delivery in vivo. In further embodiments,incorporation of one or more miRNA binding sites into an mRNA of thedisclosure can modulate accelerated blood clearance (ABC) oflipid-comprising compounds and compositions described herein.

Conversely, miRNA binding sites can be removed from polynucleotidesequences in which they naturally occur in order to increase proteinexpression in specific tissues. For example, a binding site for aspecific miRNA can be removed from a polynucleotide to improve proteinexpression in tissues or cells containing the miRNA.

Regulation of expression in multiple tissues can be accomplished throughintroduction or removal of one or more miRNA binding sites, e.g., one ormore distinct miRNA binding sites. The decision whether to remove orinsert a miRNA binding site can be made based on miRNA expressionpatterns and/or their profilings in tissues and/or cells in developmentand/or disease. Identification of miRNAs, miRNA binding sites, and theirexpression patterns and role in biology have been reported (e.g.,Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and ChereshCurr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 201226:404-413 (2011 Dec. 20. doi: 10.1038/leu.2011.356); Bartel Cell 2009136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; Gentner andNaldini, Tissue Antigens. 2012 80:393-403 and all references therein;each of which is incorporated herein by reference in its entirety).

Examples of tissues where miRNA are known to regulate mRNA, and therebyprotein expression, include, but are not limited to, liver (miR-122),muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92,miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21,miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart(miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lungepithelial cells (let-7, miR-133, miR-126).

Specifically, miRNAs are known to be differentially expressed in immunecells (also called hematopoietic cells), such as antigen presentingcells (APCs) (e.g., dendritic cells and macrophages), macrophages,monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killercells, etc. Immune cell specific miRNAs are involved in immunogenicity,autoimmunity, the immune-response to infection, inflammation, as well asunwanted immune response after gene therapy and tissue/organtransplantation. Immune cells specific miRNAs also regulate many aspectsof development, proliferation, differentiation and apoptosis ofhematopoietic cells (immune cells). For example, miR-142 and miR-146 areexclusively expressed in immune cells, particularly abundant in myeloiddendritic cells. It has been demonstrated that the immune response to apolynucleotide can be shut-off by adding miR-142 binding sites to the3′-UTR of the polynucleotide, enabling more stable gene transfer intissues and cells. miR-142 efficiently degrades exogenouspolynucleotides in antigen presenting cells and suppresses cytotoxicelimination of transduced cells (e.g., Annoni A et al., blood, 2009,114, 5152-5161; Brown B D, et al., Nat med. 2006, 12(5), 585-591; BrownB D, et al., blood, 2007, 110(13): 4144-4152, each of which isincorporated herein by reference in its entirety).

An antigen-mediated immune response can refer to an immune responsetriggered by foreign antigens, which, when entering an organism, areprocessed by the antigen presenting cells and displayed on the surfaceof the antigen presenting cells. T cells can recognize the presentedantigen and induce a cytotoxic elimination of cells that express theantigen.

Introducing a miR-142 binding site into the 5′UTR and/or 3′UTR of apolynucleotide of the present disclosure can selectively repress geneexpression in antigen presenting cells through miR-142 mediateddegradation, limiting antigen presentation in antigen presenting cells(e.g., dendritic cells) and thereby preventing antigen-mediated immuneresponse after the delivery of the polynucleotide. The polynucleotide isthen stably expressed in target tissues or cells without triggeringcytotoxic elimination.

In one embodiment, binding sites for miRNAs that are known to beexpressed in immune cells, in particular, antigen presenting cells, canbe engineered into a polynucleotide of the present disclosure tosuppress the expression of the polynucleotide in antigen presentingcells through miRNA mediated RNA degradation, subduing theantigen-mediated immune response. Expression of the polynucleotide ismaintained in non-immune cells where the immune cell specific miRNAs arenot expressed. For example, in some embodiments, to prevent animmunogenic reaction against a liver specific protein, any miR-122binding site can be removed and a miR-142 (and/or mirR-146) binding sitecan be engineered into the 5′UTR and/or 3′UTR of a polynucleotide of thepresent disclosure.

To further drive the selective degradation and suppression in APCs andmacrophage, a polynucleotide of the present disclosure can include afurther negative regulatory element in the 5′UTR and/or 3′UTR, eitheralone or in combination with miR-142 and/or miR-146 binding sites. As anon-limiting example, the further negative regulatory element is aConstitutive Decay Element (CDE).

Immune cell specific miRNAs include, but are not limited to,hsa-let-7a-2-3p, hsa-let-7a-3p, hsa-7a-5p, hsa-let-7c, hsa-let-7e-3p,hsa-let-7e-5p, hsa-let-7g-3p, hsa-let-7g-5p, hsa-let-7i-3p,hsa-let-7i-5p, miR-10a-3p, miR-10a-5p, miR-1184, hsa-let-7f-1-3p,hsa-let-7f-2-5p, hsa-let-7f-5p, miR-125b-1-3p, miR-125b-2-3p,miR-125b-5p, miR-1279, miR-130a-3p, miR-130a-5p, miR-132-3p, miR-132-5p,miR-142-3p, miR-142-5p, miR-143-3p, miR-143-5p, miR-146a-3p,miR-146a-5p, miR-146b-3p, miR-146b-5p, miR-147a, miR-147b, miR-148a-5p,miR-148a-3p, miR-150-3p, miR-150-5p, miR-151b, miR-155-3p, miR-155-5p,miR-15a-3p, miR-15a-5p, miR-15b-5p, miR-15b-3p, miR-16-1-3p,miR-16-2-3p, miR-16-5p, miR-17-5p, miR-181a-3p, miR-181a-5p,miR-181a-2-3p, miR-182-3p, miR-182-5p, miR-197-3p, miR-197-5p,miR-21-5p, miR-21-3p, miR-214-3p, miR-214-5p, miR-223-3p, miR-223-5p,miR-221-3p, miR-221-5p, miR-23b-3p, miR-23b-5p, miR-24-1-5p,miR-24-2-5p, miR-24-3p, miR-26a-1-3p, miR-26a-2-3p, miR-26a-5p,miR-26b-3p, miR-26b-5p, miR-27a-3p, miR-27a-5p, miR-27b-3p, miR-27b-5p,miR-28-3p, miR-28-5p, miR-2909, miR-29a-3p, miR-29a-5p, miR-29b-1-5p,miR-29b-2-5p, miR-29c-3p, miR-29c-5p, miR-30e-3p, miR-30e-5p,miR-331-5p, miR-339-3p, miR-339-5p, miR-345-3p, miR-345-5p, miR-346,miR-34a-3p, miR-34a-5p, miR-363-3p, miR-363-5p, miR-372, miR-377-3p,miR-377-5p, miR-493-3p, miR-493-5p, miR-542, miR-548b-5p, miR548c-5p,miR-548i, miR-548j, miR-548n, miR-574-3p, miR-598, miR-718, miR-935,miR-99a-3p, miR-99a-5p, miR-99b-3p, and miR-99b-5p. Furthermore, novelmiRNAs can be identified in immune cell through micro-arrayhybridization and microtome analysis (e.g., Jima D D et al, Blood, 2010,116:e118-e127; Vaz C et al., BMC Genomics, 2010, 11,288, the content ofeach of which is incorporated herein by reference in its entirety.)

miRNAs that are known to be expressed in the liver include, but are notlimited to, miR-107, miR-122-3p, miR-122-5p, miR-1228-3p, miR-1228-5p,miR-1249, miR-129-5p, miR-1303, miR-151a-3p, miR-151a-5p, miR-152,miR-194-3p, miR-194-5p, miR-199a-3p, miR-199a-5p, miR-199b-3p,miR-199b-5p, miR-296-5p, miR-557, miR-581, miR-939-3p, and miR-939-5p.MiRNA binding sites from any liver specific miRNA can be introduced toor removed from a polynucleotide of the present disclosure to regulateexpression of the polynucleotide in the liver. Liver specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thepresent disclosure.

miRNAs that are known to be expressed in the lung include, but are notlimited to, let-7a-2-3p, let-7a-3p, let-7a-5p, miR-126-3p, miR-126-5p,miR-127-3p, miR-127-5p, miR-130a-3p, miR-130a-5p, miR-130b-3p,miR-130b-5p, miR-133a, miR-133b, miR-134, miR-18a-3p, miR-18a-5p,miR-18b-3p, miR-18b-5p, miR-24-1-5p, miR-24-2-5p, miR-24-3p, miR-296-3p,miR-296-5p, miR-32-3p, miR-337-3p, miR-337-5p, miR-381-3p, andmiR-381-5p. miRNA binding sites from any lung specific miRNA can beintroduced to or removed from a polynucleotide of the present disclosureto regulate expression of the polynucleotide in the lung. Lung specificmiRNA binding sites can be engineered alone or further in combinationwith immune cell (e.g., APC) miRNA binding sites in a polynucleotide ofthe present disclosure.

miRNAs that are known to be expressed in the heart include, but are notlimited to, miR-1, miR-133a, miR-133b, miR-149-3p, miR-149-5p,miR-186-3p, miR-186-5p, miR-208a, miR-208b, miR-210, miR-296-3p,miR-320, miR-451a, miR-451b, miR-499a-3p, miR-499a-5p, miR-499b-3p,miR-499b-5p, miR-744-3p, miR-744-5p, miR-92b-3p, and miR-92b-5p. miRNAbinding sites from any heart specific microRNA can be introduced to orremoved from a polynucleotide of the present disclosure to regulateexpression of the polynucleotide in the heart. Heart specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thepresent disclosure.

miRNAs that are known to be expressed in the nervous system include, butare not limited to, miR-124-5p, miR-125a-3p, miR-125a-5p, miR-125b-1-3p,miR-125b-2-3p, miR-125b-5p, miR-1271-3p, miR-1271-5p, miR-128,miR-132-5p, miR-135a-3p, miR-135a-5p, miR-135b-3p, miR-135b-5p, miR-137,miR-139-5p, miR-139-3p, miR-149-3p, miR-149-5p, miR-153, miR-181c-3p,miR-181c-5p, miR-183-3p, miR-183-5p, miR-190a, miR-190b, miR-212-3p,miR-212-5p, miR-219-1-3p, miR-219-2-3p, miR-23a-3p, miR-23a-5p,miR-30a-5p, miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p,miR-30c-5p, miR-30d-3p, miR-30d-5p, miR-329, miR-342-3p, miR-3665,miR-3666, miR-380-3p, miR-380-5p, miR-383, miR-410, miR-425-3p,miR-425-5p, miR-454-3p, miR-454-5p, miR-483, miR-510, miR-516a-3p,miR-548b-5p, miR-548c-5p, miR-571, miR-7-1-3p, miR-7-2-3p, miR-7-5p,miR-802, miR-922, miR-9-3p, and miR-9-5p. miRNAs enriched in the nervoussystem further include those specifically expressed in neurons,including, but not limited to, miR-132-3p, miR-132-3p, miR-148b-3p,miR-148b-5p, miR-151a-3p, miR-151a-5p, miR-212-3p, miR-212-5p, miR-320b,miR-320e, miR-323a-3p, miR-323a-5p, miR-324-5p, miR-325, miR-326,miR-328, miR-922 and those specifically expressed in glial cells,including, but not limited to, miR-1250, miR-219-1-3p, miR-219-2-3p,miR-219-5p, miR-23a-3p, miR-23a-5p, miR-3065-3p, miR-3065-5p,miR-30e-3p, miR-30e-5p, miR-32-5p, miR-338-5p, and miR-657. miRNAbinding sites from any CNS specific miRNA can be introduced to orremoved from a polynucleotide of the present disclosure to regulateexpression of the polynucleotide in the nervous system. Nervous systemspecific miRNA binding sites can be engineered alone or further incombination with immune cell (e.g., APC) miRNA binding sites in apolynucleotide of the present disclosure.

miRNAs that are known to be expressed in the pancreas include, but arenot limited to, miR-105-3p, miR-105-5p, miR-184, miR-195-3p, miR-195-5p,miR-196a-3p, miR-196a-5p, miR-214-3p, miR-214-5p, miR-216a-3p,miR-216a-5p, miR-30a-3p, miR-33a-3p, miR-33a-5p, miR-375, miR-7-1-3p,miR-7-2-3p, miR-493-3p, miR-493-5p, and miR-944. MiRNA binding sitesfrom any pancreas specific miRNA can be introduced to or removed from apolynucleotide of the present disclosure to regulate expression of thepolynucleotide in the pancreas. Pancreas specific miRNA binding sitescan be engineered alone or further in combination with immune cell (e.g.APC) miRNA binding sites in a polynucleotide of the present disclosure.

miRNAs that are known to be expressed in the kidney include, but are notlimited to, miR-122-3p, miR-145-5p, miR-17-5p, miR-192-3p, miR-192-5p,miR-194-3p, miR-194-5p, miR-20a-3p, miR-20a-5p, miR-204-3p, miR-204-5p,miR-210, miR-216a-3p, miR-216a-5p, miR-296-3p, miR-30a-3p, miR-30a-5p,miR-30b-3p, miR-30b-5p, miR-30c-1-3p, miR-30c-2-3p, miR30c-5p,miR-324-3p, miR-335-3p, miR-335-5p, miR-363-3p, miR-363-5p, and miR-562.miRNA binding sites from any kidney specific miRNA can be introduced toor removed from a polynucleotide of the present disclosure to regulateexpression of the polynucleotide in the kidney. Kidney specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thepresent disclosure.

miRNAs that are known to be expressed in the muscle include, but are notlimited to, let-7g-3p, let-7g-5p, miR-1, miR-1286, miR-133a, miR-133b,miR-140-3p, miR-143-3p, miR-143-5p, miR-145-3p, miR-145-5p, miR-188-3p,miR-188-5p, miR-206, miR-208a, miR-208b, miR-25-3p, and miR-25-5p. MiRNAbinding sites from any muscle specific miRNA can be introduced to orremoved from a polynucleotide of the present disclosure to regulateexpression of the polynucleotide in the muscle. Muscle specific miRNAbinding sites can be engineered alone or further in combination withimmune cell (e.g., APC) miRNA binding sites in a polynucleotide of thepresent disclosure.

miRNAs are also differentially expressed in different types of cells,such as, but not limited to, endothelial cells, epithelial cells, andadipocytes.

miRNAs that are known to be expressed in endothelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-100-3p, miR-100-5p,miR-101-3p, miR-101-5p, miR-126-3p, miR-126-5p, miR-1236-3p,miR-1236-5p, miR-130a-3p, miR-130a-5p, miR-17-5p, miR-17-3p, miR-18a-3p,miR-18a-5p, miR-19a-3p, miR-19a-5p, miR-19b-1-5p, miR-19b-2-5p,miR-19b-3p, miR-20a-3p, miR-20a-5p, miR-217, miR-210, miR-21-3p,miR-21-5p, miR-221-3p, miR-221-5p, miR-222-3p, miR-222-5p, miR-23a-3p,miR-23a-5p, miR-296-5p, miR-361-3p, miR-361-5p, miR-421, miR-424-3p,miR-424-5p, miR-513a-5p, miR-92a-1-5p, miR-92a-2-5p, miR-92a-3p,miR-92b-3p, and miR-92b-5p. Many novel miRNAs are discovered inendothelial cells from deep-sequencing analysis (e.g., Voellenkle C etal., RNA, 2012, 18, 472-484, herein incorporated by reference in itsentirety). miRNA binding sites from any endothelial cell specific miRNAcan be introduced to or removed from a polynucleotide of the presentdisclosure to regulate expression of the polynucleotide in theendothelial cells.

miRNAs that are known to be expressed in epithelial cells include, butare not limited to, let-7b-3p, let-7b-5p, miR-1246, miR-200a-3p,miR-200a-5p, miR-200b-3p, miR-200b-5p, miR-200c-3p, miR-200c-5p,miR-338-3p, miR-429, miR-451a, miR-451b, miR-494, miR-802 and miR-34a,miR-34b-5p, miR-34c-5p, miR-449a, miR-449b-3p, miR-449b-5p specific inrespiratory ciliated epithelial cells, let-7 family, miR-133a, miR-133b,miR-126 specific in lung epithelial cells, miR-382-3p, miR-382-5pspecific in renal epithelial cells, and miR-762 specific in cornealepithelial cells. miRNA binding sites from any epithelial cell specificmiRNA can be introduced to or removed from a polynucleotide of thepresent disclosure to regulate expression of the polynucleotide in theepithelial cells.

In addition, a large group of miRNAs are enriched in embryonic stemcells, controlling stem cell self-renewal as well as the developmentand/or differentiation of various cell lineages, such as neural cells,cardiac, hematopoietic cells, skin cells, osteogenic cells and musclecells (e.g., Kuppusamy K T et al., Curr. Mol Med, 2013, 13(5), 757-764;Vidigal J A and Ventura A, Semin Cancer Biol. 2012, 22(5-6), 428-436;Goff L A et al., PLoS One, 2009, 4:e7192; Morin R D et al., Genome Res,2008, 18, 610-621; Yoo J K et al., Stem Cells Dev. 2012, 21(11),2049-2057, each of which is herein incorporated by reference in itsentirety). MiRNAs abundant in embryonic stem cells include, but are notlimited to, let-7a-2-3p, let-a-3p, let-7a-5p, let7d-3p, let-7d-5p,miR-103a-2-3p, miR-103a-5p, miR-106b-3p, miR-106b-5p, miR-1246,miR-1275, miR-138-1-3p, miR-138-2-3p, miR-138-5p, miR-154-3p,miR-154-5p, miR-200c-3p, miR-200c-5p, miR-290, miR-301a-3p, miR-301a-5p,miR-302a-3p, miR-302a-5p, miR-302b-3p, miR-302b-5p, miR-302c-3p,miR-302c-5p, miR-302d-3p, miR-302d-5p, miR-302e, miR-367-3p, miR-367-5p,miR-369-3p, miR-369-5p, miR-370, miR-371, miR-373, miR-380-5p,miR-423-3p, miR-423-5p, miR-486-5p, miR-520c-3p, miR-548e, miR-548f,miR-548g-3p, miR-548g-5p, miR-548i, miR-548k, miR-548l, miR-548m,miR-548n, miR-548o-3p, miR-548o-5p, miR-548p, miR-664a-3p, miR-664a-5p,miR-664b-3p, miR-664b-5p, miR-766-3p, miR-766-5p, miR-885-3p,miR-885-5p, miR-93-3p, miR-93-5p, miR-941, miR-96-3p, miR-96-5p,miR-99b-3p and miR-99b-5p. Many predicted novel miRNAs are discovered bydeep sequencing in human embryonic stem cells (e.g., Morin R D et al.,Genome Res, 2008, 18, 610-621; Goff L A et al., PLoS One, 2009, 4:e7192;Bar M et al., Stem cells, 2008, 26, 2496-2505, the content of each ofwhich is incorporated herein by reference in its entirety).

In some embodiments, miRNAs are selected based on expression andabundance in immune cells of the hematopoietic lineage, such as B cells,T cells, macrophages, dendritic cells, and cells that are known toexpress TLR7/TLR8 and/or able to secrete cytokines such as endothelialcells and platelets. In some embodiments, the miRNA set thus includesmiRs that may be responsible in part for the immunogenicity of thesecells, and such that a corresponding miR-site incorporation inpolynucleotides of the present invention (e.g., mRNAs) could lead todestabilization of the mRNA and/or suppression of translation from thesemRNAs in the specific cell type. Non-limiting representative examplesinclude miR-142, miR-144, miR-150, miR-155 and miR-223, which arespecific for many of the hematopoietic cells; miR-142, miR150, miR-16and miR-223, which are expressed in B cells; miR-223, miR-451, miR-26a,miR-16, which are expressed in progenitor hematopoietic cells; andmiR-126, which is expressed in plasmacytoid dendritic cells, plateletsand endothelial cells. For further discussion of tissue expression ofmiRs see e.g., Teruel-Montoya, R. et al. (2014) PLoS One 9:e102259;Landgraf, P. et al. (2007) Cell 129:1401-1414; Bissels, U. et al. (2009)RNA 15:2375-2384. Any one miR-site incorporation in the 3′ UTR and/or 5′UTR may mediate such effects in multiple cell types of interest (e.g.,miR-142 is abundant in both B cells and dendritic cells).

In some embodiments, it may be beneficial to target the same cell typewith multiple miRs and to incorporate binding sites to each of the 3pand 5p arm if both are abundant (e.g., both miR-142-3p and miR142-5p areabundant in hematopoietic stem cells). Thus, in certain embodiments,polynucleotides of the invention contain two or more (e.g., two, three,four or more) miR bindings sites from: (i) the group consisting ofmiR-142, miR-144, miR-150, miR-155 and miR-223 (which are expressed inmany hematopoietic cells); or (ii) the group consisting of miR-142,miR150, miR-16 and miR-223 (which are expressed in B cells); or thegroup consisting of miR-223, miR-451, miR-26a, miR-16 (which areexpressed in progenitor hematopoietic cells).

In some embodiments, it may also be beneficial to combine various miRssuch that multiple cell types of interest are targeted at the same time(e.g., miR-142 and miR-126 to target many cells of the hematopoieticlineage and endothelial cells). Thus, for example, in certainembodiments, polynucleotides of the invention comprise two or more(e.g., two, three, four or more) miRNA bindings sites, wherein: (i) atleast one of the miRs targets cells of the hematopoietic lineage (e.g.,miR-142, miR-144, miR-150, miR-155 or miR-223) and at least one of themiRs targets plasmacytoid dendritic cells, platelets or endothelialcells (e.g., miR-126); or (ii) at least one of the miRs targets B cells(e.g., miR-142, miR150, miR-16 or miR-223) and at least one of the miRstargets plasmacytoid dendritic cells, platelets or endothelial cells(e.g., miR-126); or (iii) at least one of the miRs targets progenitorhematopoietic cells (e.g., miR-223, miR-451, miR-26a or miR-16) and atleast one of the miRs targets plasmacytoid dendritic cells, platelets orendothelial cells (e.g., miR-126); or (iv) at least one of the miRstargets cells of the hematopoietic lineage (e.g., miR-142, miR-144,miR-150, miR-155 or miR-223), at least one of the miRs targets B cells(e.g., miR-142, miR150, miR-16 or miR-223) and at least one of the miRstargets plasmacytoid dendritic cells, platelets or endothelial cells(e.g., miR-126); or any other possible combination of the foregoing fourclasses of miR binding sites (i.e., those targeting the hematopoieticlineage, those targeting B cells, those targeting progenitorhematopoietic cells and/or those targeting plamacytoid dendriticcells/platelets/endothelial cells).

In one embodiment, to modulate immune responses, polynucleotides of thepresent invention can comprise one or more miRNA binding sequences thatbind to one or more miRs that are expressed in conventional immune cellsor any cell that expresses TLR7 and/or TLR8 and secrete pro-inflammatorycytokines and/or chemokines (e.g., in immune cells of peripherallymphoid organs and/or splenocytes and/or endothelial cells). It has nowbeen discovered that incorporation into an mRNA of one or more miRs thatare expressed in conventional immune cells or any cell that expressesTLR7 and/or TLR8 and secrete pro-inflammatory cytokines and/orchemokines (e.g., in immune cells of peripheral lymphoid organs and/orsplenocytes and/or endothelial cells) reduces or inhibits immune cellactivation (e.g., B cell activation, as measured by frequency ofactivated B cells) and/or cytokine production (e.g., production of IL-6,IFN-γ and/or TNFα). Furthermore, it has now been discovered thatincorporation into an mRNA of one or more miRs that are expressed inconventional immune cells or any cell that expresses TLR7 and/or TLR8and secrete pro-inflammatory cytokines and/or chemokines (e.g., inimmune cells of peripheral lymphoid organs and/or splenocytes and/orendothelial cells) can reduce or inhibit an anti-drug antibody (ADA)response against a protein of interest encoded by the mRNA.

In another embodiment, to modulate accelerated blood clearance of apolynucleotide delivered in a lipid-comprising compound or composition,polynucleotides of the invention can comprise one or more miR bindingsequences that bind to one or more miRNAs expressed in conventionalimmune cells or any cell that expresses TLR7 and/or TLR8 and secretepro-inflammatory cytokines and/or chemokines (e.g., in immune cells ofperipheral lymphoid organs and/or splenocytes and/or endothelial cells).It has now been discovered that incorporation into an mRNA of one ormore miR binding sites reduces or inhibits accelerated blood clearance(ABC) of the lipid-comprising compound or composition for use indelivering the mRNA. Furthermore, it has now been discovered thatincorporation of one or more miR binding sites into an mRNA reducesserum levels of anti-PEG anti-IgM (e.g., reduces or inhibits the acuteproduction of IgMs that recognize polyethylene glycol (PEG) by B cells)and/or reduces or inhibits proliferation and/or activation ofplasmacytoid dendritic cells following administration of alipid-comprising compound or composition comprising the mRNA.

In some embodiments, miR sequences may correspond to any known microRNAexpressed in immune cells, including but not limited to those taught inUS Publication US2005/0261218 and US Publication US2005/0059005, thecontents of which are incorporated herein by reference in theirentirety. Non-limiting examples of miRs expressed in immune cellsinclude those expressed in spleen cells, myeloid cells, dendritic cells,plasmacytoid dendritic cells, B cells, T cells and/or macrophages. Forexample, miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24 andmiR-27 are expressed in myeloid cells, miR-155 is expressed in dendriticcells, B cells and T cells, miR-146 is upregulated in macrophages uponTLR stimulation and miR-126 is expressed in plasmacytoid dendriticcells. In certain embodiments, the miR(s) is expressed abundantly orpreferentially in immune cells. For example, miR-142 (miR-142-3p and/ormiR-142-5p), miR-126 (miR-126-3p and/or miR-126-5p), miR-146 (miR-146-3pand/or miR-146-5p) and miR-155 (miR-155-3p and/or miR155-5p) areexpressed abundantly in immune cells. These microRNA sequences are knownin the art and, thus, one of ordinary skill in the art can readilydesign binding sequences or target sequences to which these microRNAswill bind based upon Watson-Crick complementarity.

Accordingly, in various embodiments, polynucleotides of the presentinvention comprise at least one microRNA binding site for a miR selectedfrom the group consisting of miR-142, miR-146, miR-155, miR-126, miR-16,miR-21, miR-223, miR-24 and miR-27. In another embodiment, the mRNAcomprises at least two miR binding sites for microRNAs expressed inimmune cells. In various embodiments, the polynucleotide of theinvention comprises 1-4, one, two, three or four miR binding sites formicroRNAs expressed in immune cells. In another embodiment, thepolynucleotide of the invention comprises three miR binding sites. ThesemiR binding sites can be for microRNAs selected from the groupconsisting of miR-142, miR-146, miR-155, miR-126, miR-16, miR-21,miR-223, miR-24, miR-27, and combinations thereof. In one embodiment,the polynucleotide of the invention comprises two or more (e.g., two,three, four) copies of the same miR binding site expressed in immunecells, e.g., two or more copies of a miR binding site selected from thegroup of miRs consisting of miR-142, miR-146, miR-155, miR-126, miR-16,miR-21, miR-223, miR-24, miR-27.

In one embodiment, the polynucleotide of the invention comprises threecopies of the same miRNA binding site. In certain embodiments, use ofthree copies of the same miR binding site can exhibit beneficialproperties as compared to use of a single miRNA binding site.Non-limiting examples of sequences for 3′ UTRs containing three miRNAbindings sites are shown in SEQ ID NO: 165 (three miR-142-3p bindingsites) and SEQ ID NO: 167 (three miR-142-5p binding sites).

In another embodiment, the polynucleotide of the invention comprises twoor more (e.g., two, three, four) copies of at least two different miRbinding sites expressed in immune cells. Non-limiting examples ofsequences of 3′ UTRs containing two or more different miR binding sitesare shown in SEQ ID NO: 135 (one miR-142-3p binding site and onemiR-126-3p binding site), SEQ ID NO: 168 (two miR-142-5p binding sitesand one miR-142-3p binding sites), and SEQ ID NO: 171 (two miR-155-5pbinding sites and one miR-142-3p binding sites).

In another embodiment, the polynucleotide of the invention comprises atleast two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-142-3p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-142-3p and miR-155 (miR-155-3p or miR-155-5p), miR-142-3p andmiR-146 (miR-146-3 or miR-146-5p), or miR-142-3p and miR-126 (miR-126-3por miR-126-5p).

In another embodiment, the polynucleotide of the invention comprises atleast two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-126-3p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-126-3p and miR-155 (miR-155-3p or miR-155-5p), miR-126-3p andmiR-146 (miR-146-3p or miR-146-5p), or miR-126-3p and miR-142(miR-142-3p or miR-142-5p).

In another embodiment, the polynucleotide of the invention comprises atleast two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-142-5p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-142-5p and miR-155 (miR-155-3p or miR-155-5p), miR-142-5p andmiR-146 (miR-146-3 or miR-146-5p), or miR-142-5p and miR-126 (miR-126-3por miR-126-5p).

In yet another embodiment, the polynucleotide of the invention comprisesat least two miR binding sites for microRNAs expressed in immune cells,wherein one of the miR binding sites is for miR-155-5p. In variousembodiments, the polynucleotide of the invention comprises binding sitesfor miR-155-5p and miR-142 (miR-142-3p or miR-142-5p), miR-155-5p andmiR-146 (miR-146-3 or miR-146-5p), or miR-155-5p and miR-126 (miR-126-3por miR-126-5p).

In one embodiment, the binding sites of embryonic stem cell specificmiRNAs can be included in or removed from the 3′UTR of a polynucleotideof the present disclosure to modulate the development and/ordifferentiation of embryonic stem cells, to inhibit the senescence ofstem cells in a degenerative condition (e.g. degenerative diseases), orto stimulate the senescence and apoptosis of stem cells in a diseasecondition (e.g. cancer stem cells).

As a non-limiting example, miRNA binding sites for miRNAs that areover-expressed in certain cancer and/or tumor cells can be removed fromthe 3′UTR of a polynucleotide of the present disclosure, restoring theexpression suppressed by the over-expressed miRNAs in cancer cells, thusameliorating the corresponsive biological function, for instance,transcription stimulation and/or repression, cell cycle arrest,apoptosis and cell death. Normal cells and tissues, wherein miRNAsexpression is not up-regulated, will remain unaffected.

miRNA can also regulate complex biological processes such asangiogenesis (e.g., miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176). In the polynucleotides of the present disclosure, miRNAbinding sites that are involved in such processes can be removed orintroduced, in order to tailor the expression of the polynucleotides tobiologically relevant cell types or relevant biological processes. Inthis context, the polynucleotides of the present disclosure are definedas auxotrophic polynucleotides.

In some embodiments, a polynucleotide of the present disclosurecomprises a miRNA binding site, wherein the miRNA binding site comprisesone or more nucleotide sequences selected from Table 3, including one ormore copies of any one or more of the miRNA binding site sequences. Insome embodiments, a polynucleotide of the present disclosure furthercomprises at least one, two, three, four, five, six, seven, eight, nine,ten, or more of the same or different miRNA binding sites selected fromTable 3, including any combination thereof.

In some embodiments, the miRNA binding site binds to miR-142 or iscomplementary to miR-142. In some embodiments, the miR-142 comprises SEQID NO:134. In some embodiments, the miRNA binding site binds tomiR-142-3p or miR-142-5p. In some embodiments, the miR-142-3p bindingsite comprises SEQ ID NO:172. In some embodiments, the miR-142-5pbinding site comprises SEQ ID NO:175. In some embodiments, the miRNAbinding site comprises a nucleotide sequence at least 80%, at least 85%,at least 90%, at least 95%, or 100% identical to SEQ ID NO:172 or SEQ IDNO:175.

In some embodiments, the miRNA binding site binds to miR-126 or iscomplementary to miR-126. In some embodiments, the miR-126 comprises SEQID NO: 139. In some embodiments, the miRNA binding site binds tomiR-126-3p or miR-126-5p. In some embodiments, the miR-126-3p bindingsite comprises SEQ ID NO: 141. In some embodiments, the miR-126-5pbinding site comprises SEQ ID NO: 143. In some embodiments, the miRNAbinding site comprises a nucleotide sequence at least 80%, at least 85%,at least 90%, at least 95%, or 100% identical to SEQ ID NO: 141 or SEQID NO: 143.

In one embodiment, the 3′ UTR comprises two miRNA binding sites, whereina first miRNA binding site binds to miR-142 and a second miRNA bindingsite binds to miR-126. In a specific embodiment, the 3′ UTR binding tomiR-142 and miR-126 comprises, consists, or consists essentially of thesequence of SEQ ID NO: 127 or 128.

TABLE 3 miR-142 and miR-142 binding sites SEQ ID NO. DescriptionSequence 134 miR-142 GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGU GUAGUGUUUCCUACUUUAUGGAUGAG UGUACUGUG 105miR-142-3p UGUAGUGUUUCCUACUUUAUGGA 172 miR-142-3pUCCAUAAAGUAGGAAACACUACA binding site 173 miR-142-5pCAUAAAGUAGAAAGCACUACU 175 miR-142-5p AGUAGUGCUUUCUACUUUAUG binding site139 miR-126 CGCUGGCGACGGGACAUUAUUACUUU UGGUACGCGCUGUGACACUUCAAACUCGUACCGUGAGUAAUAAUGCGCCGUC CACGGCA 140 miR-126-3p UCGUACCGUGAGUAAUAAUGCG141 miR-126-3p CGCAUUAUUACUCACGGUACGA binding site 142 miR-126-5pCAUUAUUACUUUUGGUACGCG 143 miR-126-5p CGCGUACCAAAAGUAAUAAUG binding site

In some embodiments, a miRNA binding site is inserted in thepolynucleotide of the present disclosure in any position of thepolynucleotide (e.g., the 5′UTR and/or 3′UTR). In some embodiments, the5′UTR comprises a miRNA binding site. In some embodiments, the 3′UTRcomprises a miRNA binding site. In some embodiments, the 5′UTR and the3′UTR comprise a miRNA binding site. The insertion site in thepolynucleotide can be anywhere in the polynucleotide as long as theinsertion of the miRNA binding site in the polynucleotide does notinterfere with the translation of a functional polypeptide in theabsence of the corresponding miRNA; and in the presence of the miRNA,the insertion of the miRNA binding site in the polynucleotide and thebinding of the miRNA binding site to the corresponding miRNA are capableof degrading the polynucleotide or preventing the translation of thepolynucleotide.

In some embodiments, a miRNA binding site is inserted in at least about30 nucleotides downstream from the stop codon of an ORF in apolynucleotide of the present disclosure comprising the ORF. In someembodiments, a miRNA binding site is inserted in at least about 10nucleotides, at least about 15 nucleotides, at least about 20nucleotides, at least about 25 nucleotides, at least about 30nucleotides, at least about 35 nucleotides, at least about 40nucleotides, at least about 45 nucleotides, at least about 50nucleotides, at least about 55 nucleotides, at least about 60nucleotides, at least about 65 nucleotides, at least about 70nucleotides, at least about 75 nucleotides, at least about 80nucleotides, at least about 85 nucleotides, at least about 90nucleotides, at least about 95 nucleotides, or at least about 100nucleotides downstream from the stop codon of an ORF in a polynucleotideof the present disclosure. In some embodiments, a miRNA binding site isinserted in about 10 nucleotides to about 100 nucleotides, about 20nucleotides to about 90 nucleotides, about 30 nucleotides to about 80nucleotides, about 40 nucleotides to about 70 nucleotides, about 50nucleotides to about 60 nucleotides, about 45 nucleotides to about 65nucleotides downstream from the stop codon of an ORF in a polynucleotideof the present disclosure.

In some embodiments, a miRNA binding site is inserted within the 3′ UTRimmediately following the stop codon of the coding region within thepolynucleotide of the invention, e.g., mRNA. In some embodiments, ifthere are multiple copies of a stop codon in the construct, a miRNAbinding site is inserted immediately following the final stop codon. Insome embodiments, a miRNA binding site is inserted further downstream ofthe stop codon, in which case there are 3′ UTR bases between the stopcodon and the miR binding site(s). In some embodiments, threenon-limiting examples of possible insertion sites for a miR in a 3′ UTRare shown in SEQ ID NOs: 127, 128, and 174, which show a 3′ UTR sequencewith a miR-142-3p site inserted in one of three different possibleinsertion sites, respectively, within the 3′ UTR.

In some embodiments, one or more miRNA binding sites can be positionedwithin the 5′ UTR at one or more possible insertion sites. For example,three non-limiting examples of possible insertion sites for a miR in a5′ UTR are shown in SEQ ID NOs: 176, 177, and 178, which show a 5′ UTRsequence with a miR-142-3p site inserted into one of three differentpossible insertion sites, respectively, within the 5′ UTR.

In one embodiment, a codon optimized open reading frame encoding apolypeptide of interest comprises a stop codon and the at least onemicroRNA binding site is located within the 3′ UTR 1-100 nucleotidesafter the stop codon. In one embodiment, the codon optimized openreading frame encoding the polypeptide of interest comprises a stopcodon and the at least one microRNA binding site for a miR expressed inimmune cells is located within the 3′ UTR 30-50 nucleotides after thestop codon. In another embodiment, the codon optimized open readingframe encoding the polypeptide of interest comprises a stop codon andthe at least one microRNA binding site for a miR expressed in immunecells is located within the 3′ UTR at least 50 nucleotides after thestop codon. In other embodiments, the codon optimized open reading frameencoding the polypeptide of interest comprises a stop codon and the atleast one microRNA binding site for a miR expressed in immune cells islocated within the 3′ UTR immediately after the stop codon, or withinthe 3′ UTR 15-20 nucleotides after the stop codon or within the 3′ UTR70-80 nucleotides after the stop codon. In other embodiments, the 3′ UTRcomprises more than one miRNA binding site (e.g., 2-4 miRNA bindingsites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or30-50 nucleotides in length) between each miRNA binding site. In anotherembodiment, the 3′ UTR comprises a spacer region between the end of themiRNA binding site(s) and the poly A tail nucleotides. For example, aspacer region of 10-100, 20-70 or 30-50 nucleotides in length can besituated between the end of the miRNA binding site(s) and the beginningof the poly A tail.

In one embodiment, a codon optimized open reading frame encoding apolypeptide of interest comprises a start codon and the at least onemicroRNA binding site is located within the 5′ UTR 1-100 nucleotidesbefore (upstream of) the start codon. In one embodiment, the codonoptimized open reading frame encoding the polypeptide of interestcomprises a start codon and the at least one microRNA binding site for amiR expressed in immune cells is located within the 5′ UTR 10-50nucleotides before (upstream of) the start codon. In another embodiment,the codon optimized open reading frame encoding the polypeptide ofinterest comprises a start codon and the at least one microRNA bindingsite for a miR expressed in immune cells is located within the 5′ UTR atleast 25 nucleotides before (upstream of) the start codon. In otherembodiments, the codon optimized open reading frame encoding thepolypeptide of interest comprises a start codon and the at least onemicroRNA binding site for a miR expressed in immune cells is locatedwithin the 5′ UTR immediately before the start codon, or within the 5′UTR 15-20 nucleotides before the start codon or within the 5′ UTR 70-80nucleotides before the start codon. In other embodiments, the 5′ UTRcomprises more than one miRNA binding site (e.g., 2-4 miRNA bindingsites), wherein there can be a spacer region (e.g., of 10-100, 20-70 or30-50 nucleotides in length) between each miRNA binding site.

In one embodiment, the 3′ UTR comprises more than one stop codon,wherein at least one miRNA binding site is positioned downstream of thestop codons. For example, a 3′ UTR can comprise 1, 2 or 3 stop codons.Non-limiting examples of triple stop codons that can be used include:UGAUAAUAG (SEQ ID NO:195), UGAUAGUAA (SEQ ID NO:196), UAAUGAUAG (SEQ IDNO:197), UGAUAAUAA (SEQ ID NO:198), UGAUAGUAG (SEQ ID NO:199), UAAUGAUGA(SEQ ID NO:200), UAAUAGUAG (SEQ ID NO:201), UGAUGAUGA (SEQ ID NO:202),UAAUAAUAA (SEQ ID NO:203), and UAGUAGUAG (SEQ ID NO:204). Within a 3′UTR, for example, 1, 2, 3 or 4 miRNA binding sites, e.g., miR-142-3pbinding sites, can be positioned immediately adjacent to the stopcodon(s) or at any number of nucleotides downstream of the final stopcodon. When the 3′ UTR comprises multiple miRNA binding sites, thesebinding sites can be positioned directly next to each other in theconstruct (i.e., one after the other) or, alternatively, spacernucleotides can be positioned between each binding site.

In one embodiment, the 3′ UTR comprises three stop codons with a singlemiR-142-3p binding site located downstream of the 3rd stop codon.Non-limiting examples of sequences of 3′ UTR having three stop codonsand a single miR-142-3p binding site located at different positionsdownstream of the final stop codon are shown in SEQ ID NOs: 132, 127,128, and 174.

TABLE 4 5′ UTRs, 3′UTRs, miR sequences, and miR binding sites SEQ  IDNO: Sequence 144 GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (3′ UTR with miR 142-3p binding site) 172UCCAUAAAGUAGGAAACACUACA (miR 142-3p binding site) 105UGUAGUGUUUCCUACUUUAUGGA (miR 142-3p sequence) 173 CAUAAAGUAGAAAGCACUACU(miR 142-5p sequence) 145 CCUCUGAAAUUCAGUUCUUCAG (miR 146-3p sequence)146 UGAGAACUGAAUUCCAUGGGUU (miR 146-5p sequence) 147CUCCUACAUAUUAGCAUUAACA (miR 155-3p sequence) 148 UUAAUGCUAAUCGUGAUAGGGGU(miR 155-5p sequence) 140 UCGUACCGUGAGUAAUAAUGCG (miR 126-3p sequence)142 CAUUAUUACUUUUGGUACGCG (miR 126-5p sequence) 149CCAGUAUUAACUGUGCUGCUGA (miR 16-3p sequence) 150 UAGCAGCACGUAAAUAUUGGCG(miR 16-5p sequence) 151 CAACACCAGUCGAUGGGCUGU (miR 21-3p sequence) 152UAGCUUAUCAGACUGAUGUUGA (miR 21-5p sequence) 153 UGUCAGUUUGUCAAAUACCCCA(miR 223-3p sequence) 154 CGUGUAUUUGACAAGCUGAGUU (miR 223-5p sequence)155 UGGCUCAGUUCAGCAGGAACAG (miR 24-3p sequence) 156UGCCUACUGAGCUGAUAUCAGU (miR 24-5p sequence) 157 UUCACAGUGGCUAAGUUCCGC(miR 27-3p sequence) 158 AGGGCUUAGCUGCUUGUGAGCA (miR 27-5p sequence) 141CGCAUUAUUACUCACGGUACGA (miR 126-3p binding site) 159UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC

(3′ UTR with miR 126-3p binding site) 160UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGC(3′ UTR, no miR binding sites) 132UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site) 135 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG

UGGGCGGC (3′ UTR with miR 142-3p and miR 126-3p binding sites variant 1)163 UUAAUGCUAAUUGUGAUAGGGGU (miR 155-5p sequence) 164ACCCCUAUCACAAUUAGCAUUAA (miR 155-5p binding site) 165 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with 3 miR 142-3p binding sites) 166UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC

(3′ UTR with miR 142-5p binding site) 167

CUUUGAAUAAAGUCUGAGUGGGCGGC (3′ UTR with 3 miR 142-5p binding sites) 168

UUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCC

GUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with 2 miR 142-5p binding sites and 1 miR 142-3p binding site)169 UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCC

(3′ UTR with miR 155-5p binding site) 170

(3′ UTR with 3 miR 155-5p binding sites) 171

GCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGC

(3′ UTR with 2 miR 155-5p binding sites and 1 miR 142-3p  binding site)127 UGAUAAUAG UCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site, P1 insertion) 128UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site, P2 insertion) 174UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site, P3 insertion) 175AGUAGUGCUUUCUACUUUAUG (miR-142-5p binding site) 134GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG (miR-142) 108GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (5′ UTR) 176GGGAAAUAAGAGUCCAUAAAGUAGGAAACACUACAAGAAAAGAAGAGUAAGA AGAAAUAUAAGAGCCACC(5′ UTR with miR 142-3p binding site at position p1) 177GGGAAAUAAGAGAGAAAAGAAGAGUAAUCCAUAAAGUAGGAAACACUACAGA AGAAAUAUAAGAGCCACC(5′ UTR with miR 142-3p binding site at position p2) 178GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAUCCAUAAAGUAGG AAACACUACAGAGCCACC(5′ UTR with miR 142-3p binding site at position p3) 180

UUUGAAUAAAGUCUGAGUGGGCGGC (3′ UTR with 3 miR 142-5p binding sites) 129UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUCCAUAAAGUAGGAAACACUACAUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR including miR 142-3p binding site) 130UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGUCCAUAAAGUAGGAAACACUACACCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR including miR 142-3p binding site) 131UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCUCCAUAAAGUAGGAAACACUACACUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR including miR 142-3p binding site) 133UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUUCCAUAAAGUAGGAAACACUACACUGAGUGGGCGGC(3′ UTR including miR 142-3p binding site) 136 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCG

UGGGCGGC (3′ UTR with miR 142-3p and miR 126-3p binding sites variant 2)14 UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAA GUCUGAGUGGGCGGC(3′ UTR, no miR binding sites variant 2) 137UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site variant 3) 187UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCC

(3′ UTR with miR 126-3p binding site variant 3) 188 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCUCCAUAAAGUAGGAAACACUACAGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with 3 miR 142-3p binding sites variant 2) 189 UGAUAAUAGUCCAUAAAGUAGGAAACACUACAGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site, P1 insertion variant 2) 190UGAUAAUAGGCUGGAGCCUCGGUGGCUCCAUAAAGUAGGAAACACUACACUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site, P2 insertion variant 2) 191UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(3′ UTR with miR 142-3p binding site, P3 insertion variant 2) 192UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCC

(3′ UTR with miR 155-5p binding site variant 2) 193

(3′ UTR with 3 miR 155-5p binding sites variant 2) 194

GCUUCUUGCCCCUUGGGCCUCCAUAAAGUAGGAAACACUACAUCCCCCCAGC

(3′ UTR with 2 miR 155-5p binding sites and 1 miR 142-3pbinding site variant 2) Stop codon = bold miR 142-3p binding site= underline miR 126-3p binding site = bold underline miR 155-5p bindingsite = shaded miR 142-5p binding site = shaded and bold underline

In one embodiment, the polynucleotide of the invention comprises a 5′UTR, a codon optimized open reading frame encoding a polypeptide ofinterest, a 3′ UTR comprising the at least one miRNA binding site for amiR expressed in immune cells, and a 3′ tailing region of linkednucleosides. In various embodiments, the 3′ UTR comprises 1-4, at leasttwo, one, two, three or four miRNA binding sites for miRs expressed inimmune cells, preferably abundantly or preferentially expressed inimmune cells.

In one embodiment, the at least one miRNA expressed in immune cells is amiR-142-3p microRNA binding site. In one embodiment, the miR-142-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO:172. Inone embodiment, the 3′ UTR of the mRNA comprising the miR-142-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 144.

In one embodiment, the at least one miRNA expressed in immune cells is amiR-126 microRNA binding site. In one embodiment, the miR-126 bindingsite is a miR-126-3p binding site. In one embodiment, the miR-126-3pmicroRNA binding site comprises the sequence shown in SEQ ID NO: 141. Inone embodiment, the 3′ UTR of the mRNA of the invention comprising themiR-126-3p microRNA binding site comprises the sequence shown in SEQ IDNO: 159.

Non-limiting exemplary sequences for miRs to which a microRNA bindingsite(s) of the disclosure can bind include the following: miR-142-3p(SEQ ID NO:172), miR-142-5p (SEQ ID NO: 175), miR-146-3p (SEQ ID NO:145), miR-146-5p (SEQ ID NO: 146), miR-155-3p (SEQ ID NO: 147),miR-155-5p (SEQ ID NO: 148), miR-126-3p (SEQ ID NO: 140), miR-126-5p(SEQ ID NO: 142), miR-16-3p (SEQ ID NO: 149), miR-16-5p (SEQ ID NO:150), miR-21-3p (SEQ ID NO: 151), miR-21-5p (SEQ ID NO: 152), miR-223-3p(SEQ ID NO: 153), miR-223-5p (SEQ ID NO: 154), miR-24-3p (SEQ ID NO:155), miR-24-5p (SEQ ID NO: 156), miR-27-3p (SEQ ID NO: 157) andmiR-27-5p (SEQ ID NO: 158). Other suitable miR sequences expressed inimmune cells (e.g., abundantly or preferentially expressed in immunecells) are known and available in the art, for example at the Universityof Manchester's microRNA database, miRBase. Sites that bind any of theaforementioned miRs can be designed based on Watson-Crickcomplementarity to the miR, typically 100% complementarity to the miR,and inserted into an mRNA construct of the disclosure as describedherein.

In another embodiment, a polynucleotide of the present invention (e.g.,and mRNA, e.g., the 3′ UTR thereof) can comprise at least one miRNAbinding site to thereby reduce or inhibit accelerated blood clearance,for example by reducing or inhibiting production of IgMs, e.g., againstPEG, by B cells and/or reducing or inhibiting proliferation and/oractivation of pDCs, and can comprise at least one miRNA binding site formodulating tissue expression of an encoded protein of interest.

miRNA gene regulation can be influenced by the sequence surrounding themiRNA such as, but not limited to, the species of the surroundingsequence, the type of sequence (e.g., heterologous, homologous,exogenous, endogenous, or artificial), regulatory elements in thesurrounding sequence and/or structural elements in the surroundingsequence. The miRNA can be influenced by the 5′UTR and/or 3′UTR. As anon-limiting example, a non-human 3′UTR can increase the regulatoryeffect of the miRNA sequence on the expression of a polypeptide ofinterest compared to a human 3′UTR of the same sequence type.

In one embodiment, other regulatory elements and/or structural elementsof the 5′UTR can influence miRNA mediated gene regulation. One exampleof a regulatory element and/or structural element is a structured IRES(Internal Ribosome Entry Site) in the 5′UTR, which is necessary for thebinding of translational elongation factors to initiate proteintranslation. EIF4A2 binding to this secondarily structured element inthe 5′-UTR is necessary for miRNA mediated gene expression (Meijer H Aet al., Science, 2013, 340, 82-85, herein incorporated by reference inits entirety). The polynucleotides of the present disclosure can furtherinclude this structured 5′UTR in order to enhance microRNA mediated generegulation.

At least one miRNA binding site can be engineered into the 3′UTR of apolynucleotide of the present disclosure. In this context, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten, or more miRNAbinding sites can be engineered into a 3′UTR of a polynucleotide of thepresent disclosure. For example, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to6, 1 to 5, 1 to 4, 1 to 3, 2, or 1 miRNA binding sites can be engineeredinto the 3′UTR of a polynucleotide of the present disclosure. In oneembodiment, miRNA binding sites incorporated into a polynucleotide ofthe present disclosure can be the same or can be different miRNA sites.A combination of different miRNA binding sites incorporated into apolynucleotide of the present disclosure can include combinations inwhich more than one copy of any of the different miRNA sites areincorporated. In another embodiment, miRNA binding sites incorporatedinto a polynucleotide of the present disclosure can target the same ordifferent tissues in the body. As a non-limiting example, through theintroduction of tissue-, cell-type-, or disease-specific miRNA bindingsites in the 3′-UTR of a polynucleotide of the present disclosure, thedegree of expression in specific cell types (e.g., hepatocytes, myeloidcells, endothelial cells, cancer cells, etc.) can be reduced.

In one embodiment, a miRNA binding site can be engineered near the 5′terminus of the 3′UTR, about halfway between the 5′ terminus and 3′terminus of the 3′UTR and/or near the 3′ terminus of the 3′UTR in apolynucleotide of the present disclosure. As a non-limiting example, amiRNA binding site can be engineered near the 5′ terminus of the 3′UTRand about halfway between the 5′ terminus and 3′ terminus of the 3′UTR.As another non-limiting example, a miRNA binding site can be engineerednear the 3′ terminus of the 3′UTR and about halfway between the 5′terminus and 3′ terminus of the 3′UTR. As yet another non-limitingexample, a miRNA binding site can be engineered near the 5′ terminus ofthe 3′UTR and near the 3′ terminus of the 3′UTR.

In another embodiment, a 3′UTR can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 miRNA binding sites. The miRNA binding sites can be complementaryto a miRNA, miRNA seed sequence, and/or miRNA sequences flanking theseed sequence.

In one embodiment, a polynucleotide of the present disclosure can beengineered to include more than one miRNA site expressed in differenttissues or different cell types of a subject. As a non-limiting example,a polynucleotide of the present disclosure can be engineered to includemiR-192 and miR-122 to regulate expression of the polynucleotide in theliver and kidneys of a subject. In another embodiment, a polynucleotideof the present disclosure can be engineered to include more than onemiRNA site for the same tissue.

In some embodiments, the expression of a polynucleotide of the presentdisclosure can be controlled by incorporating at least one miR bindingsite in the polynucleotide and formulating the polynucleotide foradministration. As a non-limiting example, a polynucleotide of thepresent disclosure can be targeted to a tissue or cell by incorporatinga miRNA binding site and formulating the polynucleotide in a lipidnanoparticle comprising an ionizable lipid, including any of the lipidsdescribed herein.

A polynucleotide of the present disclosure can be engineered for moretargeted expression in specific tissues, cell types, or biologicalconditions based on the expression patterns of miRNAs in the differenttissues, cell types, or biological conditions. Through introduction oftissue-specific miRNA binding sites, a polynucleotide of the presentdisclosure can be designed for optimal protein expression in a tissue orcell, or in the context of a biological condition.

In some embodiments, a polynucleotide of the present disclosure can bedesigned to incorporate miRNA binding sites that either have 100%identity to known miRNA seed sequences or have less than 100% identityto miRNA seed sequences. In some embodiments, a polynucleotide of thepresent disclosure can be designed to incorporate miRNA binding sitesthat have at least: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, or 99% identity to known miRNA seed sequences. The miRNA seedsequence can be partially mutated to decrease miRNA binding affinity andas such result in reduced downmodulation of the polynucleotide. Inessence, the degree of match or mis-match between the miRNA binding siteand the miRNA seed can act as a rheostat to more finely tune the abilityof the miRNA to modulate protein expression. In addition, mutation inthe non-seed region of a miRNA binding site can also impact the abilityof a miRNA to modulate protein expression.

In one embodiment, a miRNA sequence can be incorporated into the loop ofa stem loop.

In another embodiment, a miRNA seed sequence can be incorporated in theloop of a stem loop and a miRNA binding site can be incorporated intothe 5′ or 3′ stem of the stem loop.

In one embodiment the miRNA sequence in the 5′UTR can be used tostabilize a polynucleotide of the present disclosure described herein.

In another embodiment, a miRNA sequence in the 5′UTR of a polynucleotideof the present disclosure can be used to decrease the accessibility ofthe site of translation initiation such as, but not limited to a startcodon. See, e.g., Matsuda et al., PLoS One. 2010 11(5):e15057;incorporated herein by reference in its entirety, which used antisenselocked nucleic acid (LNA) oligonucleotides and exon-junction complexes(EJCs) around a start codon (−4 to +37 where the A of the AUG codons is+1) in order to decrease the accessibility to the first start codon(AUG). Matsuda showed that altering the sequence around the start codonwith an LNA or EJC affected the efficiency, length and structuralstability of a polynucleotide. A polynucleotide of the presentdisclosure can comprise a miRNA sequence, instead of the LNA or EJCsequence described by Matsuda et al, near the site of translationinitiation in order to decrease the accessibility to the site oftranslation initiation. The site of translation initiation can be priorto, after or within the miRNA sequence. As a non-limiting example, thesite of translation initiation can be located within a miRNA sequencesuch as a seed sequence or binding site. As another non-limitingexample, the site of translation initiation can be located within amiR-122 sequence such as the seed sequence or the mir-122 binding site.

In some embodiments, a polynucleotide of the present disclosure caninclude at least one miRNA in order to dampen the antigen presentationby antigen presenting cells. The miRNA can be the complete miRNAsequence, the miRNA seed sequence, the miRNA sequence without the seed,or a combination thereof. As a non-limiting example, a miRNAincorporated into a polynucleotide of the present disclosure can bespecific to the hematopoietic system. As another non-limiting example, amiRNA incorporated into a polynucleotide of the present disclosure todampen antigen presentation is miR-142-3p.

In some embodiments, a polynucleotide of the present disclosure caninclude at least one miRNA in order to dampen expression of the encodedpolypeptide in a tissue or cell of interest. As a non-limiting example,a polynucleotide of the present disclosure can include at least onemiR-122 binding site in order to dampen expression of an encodedpolypeptide of interest in the liver. As another non-limiting example apolynucleotide of the present disclosure can include at least onemiR-142-3p binding site, miR-142-3p seed sequence, miR-142-3p bindingsite without the seed, miR-142-5p binding site, miR-142-5p seedsequence, miR-142-5p binding site without the seed, miR-146 bindingsite, miR-146 seed sequence and/or miR-146 binding site without the seedsequence.

In some embodiments, a polynucleotide of the present disclosure cancomprise at least one miRNA binding site in the 3′UTR in order toselectively degrade mRNA therapeutics in the immune cells to subdueunwanted immunogenic reactions caused by therapeutic delivery. As anon-limiting example, the miRNA binding site can make a polynucleotideof the present disclosure more unstable in antigen presenting cells.Non-limiting examples of these miRNAs include mir-142-5p, mir-142-3p,mir-146a-5p, and mir-146-3p.

In one embodiment, a polynucleotide of the present disclosure comprisesat least one miRNA sequence in a region of the polynucleotide that caninteract with a RNA binding protein.

In some embodiments, the polynucleotide of the present disclosure (e.g.,a RNA, e.g., an mRNA) comprising (i) a sequence-optimized nucleotidesequence (e.g., an ORF) encoding an anti-CHIKV antibody polypeptide(e.g., a heavy chain or light chain, functional fragment, or variantthereof) and (ii) a miRNA binding site (e.g., a miRNA binding site thatbinds to miR-142) and/or a miRNA binding site that binds to miR-126.

In some embodiments, the polynucleotide of the present disclosure (e.g.,a RNA, e.g., an mRNA) comprises a sequence-optimized nucleotide sequence(e.g., an ORF) encoding an anti-CHIKV antibody polypeptide (e.g., fulllength antibody polypeptide (e.g., a heavy chain or a light chain),scFv, functional fragment, or variant thereof), wherein thepolynucleotide comprises N1-methylpseudouridines. In some embodiments,the polynucleotide further comprises a 5′ UTR having SEQ ID NO. 13 and a3′UTR having SEQ ID NO. 14. In some embodiments, the polynucleotidedisclosed herein is formulated with a delivery agent, e.g., a lipidnanoparticle comprised of an PEG-lipid of Compound I and an ionizablelipid of Compound II or Compound VI.

12. 3′ UTRs

In certain embodiments, a polynucleotide of the present disclosure(e.g., a polynucleotide comprising a nucleotide sequence encoding ananti-CHIKV antibody polypeptide of the present disclosure) furthercomprises a 3′ UTR.

3′-UTR is the section of mRNA that immediately follows the translationtermination codon and often contains regulatory regions thatpost-transcriptionally influence gene expression. Regulatory regionswithin the 3′-UTR can influence polyadenylation, translation efficiency,localization, and stability of the mRNA. In one embodiment, the 3′-UTRuseful for the present disclosure comprises a binding site forregulatory proteins or microRNAs.

In certain embodiments, the 3′ UTR useful for the polynucleotides of theinvention comprises a 3′ UTR selected from the group consisting of SEQID NOs:14 and 127-138 or any combination thereof. In some embodiments,the 3′ UTR comprises a nucleic acid sequence of SEQ ID NO: 14.

In certain embodiments, the 3′ UTR sequence useful for the inventioncomprises a nucleotide sequence at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or about 100% identical to a sequence selected from the group consistingof 3′ UTR sequences selected from the group consisting of SEQ ID NOs: 14and 127-138 or any combination thereof. In certain embodiments, the 3′UTR sequence useful for the invention comprises a nucleotide sequence atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 96%, at least about 97%, atleast about 98%, at least about 99%, or about 100% identical to SEQ IDNO: 14.

13. Regions having a 5′ Cap

The present disclosure also includes a polynucleotide that comprisesboth a 5′ Cap and a polynucleotide of the present disclosure (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide).

The 5′ cap structure of a natural mRNA is involved in nuclear export,increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP),which is responsible for mRNA stability in the cell and translationcompetency through the association of CBP with poly(A) binding proteinto form the mature cyclic mRNA species. The cap further assists theremoval of 5′ proximal introns during mRNA splicing.

Endogenous mRNA molecules can be 5′-end capped generating a5′-ppp-5′-triphosphate linkage between a terminal guanosine cap residueand the 5′-terminal transcribed sense nucleotide of the mRNA molecule.This 5′-guanylate cap can then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and/oranteterminal transcribed nucleotides of the 5′ end of the mRNA canoptionally also be 2′-O-methylated. 5′-decapping through hydrolysis andcleavage of the guanylate cap structure can target a nucleic acidmolecule, such as an mRNA molecule, for degradation.

In some embodiments, the polynucleotides of the present disclosure(e.g., a polynucleotide comprising a nucleotide sequence encoding ananti-CHIKV antibody polypeptide) incorporate a cap moiety.

In some embodiments, polynucleotides of the present disclosure (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) comprise a non-hydrolyzable cap structurepreventing decapping and thus increasing mRNA half-life. Because capstructure hydrolysis requires cleavage of 5′-ppp-5′ phosphorodiesterlinkages, modified nucleotides can be used during the capping reaction.For example, a Vaccinia Capping Enzyme from New England Biolabs(Ipswich, MA) can be used with u-thio-guanosine nucleotides according tothe manufacturer's instructions to create a phosphorothioate linkage inthe 5′-ppp-5′ cap. Additional modified guanosine nucleotides can be usedsuch as α-methyl-phosphonate and seleno-phosphate nucleotides.

Additional modifications include, but are not limited to,2′-O-methylation of the ribose sugars of 5′-terminal and/or5′-anteterminal nucleotides of the polynucleotide (as mentioned above)on the 2′-hydroxyl group of the sugar ring. Multiple distinct 5′-capstructures can be used to generate the 5′-cap of a nucleic acidmolecule, such as a polynucleotide that functions as an mRNA molecule.Cap analogs, which herein are also referred to as synthetic cap analogs,chemical caps, chemical cap analogs, or structural or functional capanalogs, differ from natural (i.e., endogenous, wild-type orphysiological) 5′-caps in their chemical structure, while retaining capfunction. Cap analogs can be chemically (i.e., non-enzymatically) orenzymatically synthesized and/or linked to the polynucleotides of thepresent disclosure.

For example, the Anti-Reverse Cap Analog (ARCA) cap contains twoguanines linked by a 5′-5′-triphosphate group, wherein one guaninecontains an N7 methyl group as well as a 3′-O-methyl group (i.e.,N7,3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine (m⁷G-3′mppp-G;which can equivalently be designated 3′ O-Me-m7G(5′)ppp(5′)G). The 3′-0atom of the other, unmodified, guanine becomes linked to the 5′-terminalnucleotide of the capped polynucleotide. The N7- and 3′-O-methlyatedguanine provides the terminal moiety of the capped polynucleotide.

Another exemplary cap is mCAP, which is similar to ARCA but has a2′-O-methyl group on guanosine (i.e.,N7,2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, m⁷Gm-ppp-G).

In some embodiments, the cap is a dinucleotide cap analog. As anon-limiting example, the dinucleotide cap analog can be modified atdifferent phosphate positions with a boranophosphate group or aphophoroselenoate group such as the dinucleotide cap analogs describedin U.S. Pat. No. 8,519,110, the contents of which are hereinincorporated by reference in its entirety.

In another embodiment, the cap is a cap analog is aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analogknown in the art and/or described herein. Non-limiting examples of aN7-(4-chlorophenoxyethyl) substituted dinucleotide form of a cap analoginclude a N7-(4-chlorophenoxyethyl)-G(5′)ppp(5′)G and aN7-(4-chlorophenoxyethyl)-m^(3,-O)G(5′)ppp(5′)G cap analog (See, e.g.,the various cap analogs and the methods of synthesizing cap analogsdescribed in Kore et al. Bioorganic & Medicinal Chemistry 201321:4570-4574; the contents of which are herein incorporated by referencein its entirety). In another embodiment, a cap analog of the presentdisclosure is a 4-chloro/bromophenoxyethyl analog.

While cap analogs allow for the concomitant capping of a polynucleotideor a region thereof, in an in vitro transcription reaction, up to 20% oftranscripts can remain uncapped. This, as well as the structuraldifferences of a cap analog from an endogenous 5′-cap structures ofnucleic acids produced by the endogenous, cellular transcriptionmachinery, can lead to reduced translational competency and reducedcellular stability.

Polynucleotides of the present disclosure (e.g., a polynucleotidecomprising a nucleotide sequence encoding an anti-CHIKV antibodypolypeptide) can also be capped post-manufacture (whether IVT orchemical synthesis), using enzymes, in order to generate more authentic5′-cap structures. As used herein, the phrase “more authentic” refers toa feature that closely mirrors or mimics, either structurally orfunctionally, an endogenous or wild type feature. That is, a “moreauthentic” feature is better representative of an endogenous, wild-type,natural or physiological cellular function and/or structure as comparedto synthetic features or analogs, etc., of the prior art, or whichoutperforms the corresponding endogenous, wild-type, natural orphysiological feature in one or more respects. Non-limiting examples ofmore authentic 5′cap structures of the present disclosure are thosethat, among other things, have enhanced binding of cap binding proteins,increased half-life, reduced susceptibility to 5′ endonucleases and/orreduced 5′decapping, as compared to synthetic 5′cap structures known inthe art (or to a wild-type, natural or physiological 5′cap structure).For example, recombinant Vaccinia Virus Capping Enzyme and recombinant2′-O-methyltransferase enzyme can create a canonical 5′-5′-triphosphatelinkage between the 5′-terminal nucleotide of a polynucleotide and aguanine cap nucleotide wherein the cap guanine contains an N7methylation and the 5′-terminal nucleotide of the mRNA contains a2′-O-methyl. Such a structure is termed the Cap1 structure. This capresults in a higher translational-competency and cellular stability anda reduced activation of cellular pro-inflammatory cytokines, ascompared, e.g., to other 5′cap analog structures known in the art. Capstructures include, but are not limited to, 7mG(5′)ppp(5′)N, pN2p (cap0), 7mG(5′)ppp(5′)NlmpNp (cap 1), and 7mG(5′)-ppp(5′)NlmpN2mp (cap 2).

As a non-limiting example, capping chimeric polynucleotidespost-manufacture can be more efficient as nearly 100% of the chimericpolynucleotides can be capped. This is in contrast to ˜80% when a capanalog is linked to a chimeric polynucleotide in the course of an invitro transcription reaction.

According to the present disclosure, 5′ terminal caps can includeendogenous caps or cap analogs. According to the present disclosure, a5′ terminal cap can comprise a guanine analog. Useful guanine analogsinclude, but are not limited to, inosine, N1-methyl-guanosine,2′fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

14. Poly-A Tails

In some embodiments, the polynucleotides of the present disclosure(e.g., a polynucleotide comprising a nucleotide sequence encoding ananti-CHIKV antibody polypeptide) further comprise a poly-A tail. Infurther embodiments, terminal groups on the poly-A tail can beincorporated for stabilization. In other embodiments, a poly-A tailcomprises des-3′ hydroxyl tails.

During RNA processing, a long chain of adenine nucleotides (poly-A tail)can be added to a polynucleotide such as an mRNA molecule in order toincrease stability. Immediately after transcription, the 3′ end of thetranscript can be cleaved to free a 3′ hydroxyl. Then poly-A polymeraseadds a chain of adenine nucleotides to the RNA. The process, calledpolyadenylation, adds a poly-A tail that can be between, for example,approximately 80 to approximately 250 residues long, includingapproximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,200, 210, 220, 230, 240 or 250 residues long.

Poly A tails can also be added after the construct is exported from thenucleus.

According to the present disclosure, terminal groups on the poly A tailcan be incorporated for stabilization. Polynucleotides of the presentdisclosure can include des-3′ hydroxyl tails. They can also includestructural moieties or 2′-Omethyl modifications as taught by Junjie Li,et al. (Current Biology, Vol. 15, 1501-1507, Aug. 23, 2005, the contentsof which are incorporated herein by reference in its entirety).

The polynucleotides of the present disclosure can be designed to encodetranscripts with alternative polyA tail structures including histonemRNA. According to Norbury, “Terminal uridylation has also been detectedon human replication-dependent histone mRNAs. The turnover of thesemRNAs is thought to be important for the prevention of potentially toxichistone accumulation following the completion or inhibition ofchromosomal DNA replication. These mRNAs are distinguished by their lackof a 3′ poly(A) tail, the function of which is instead assumed by astable stem-loop structure and its cognate stem-loop binding protein(SLBP); the latter carries out the same functions as those of PABP onpolyadenylated mRNAs” (Norbury, “Cytoplasmic RNA: a case of the tailwagging the dog,” Nature Reviews Molecular Cell Biology; AOP, publishedonline 29 Aug. 2013; doi:10.1038/nrm3645) the contents of which areincorporated herein by reference in its entirety.

Unique poly-A tail lengths provide certain advantages to thepolynucleotides of the present disclosure. Generally, the length of apoly-A tail, when present, is greater than 30 nucleotides in length. Inanother embodiment, the poly-A tail is greater than 35 nucleotides inlength (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600,700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700,1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides).

In some embodiments, the polynucleotide or region thereof includes fromabout 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000,from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100,from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750,from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500,and from 2,500 to 3,000).

In some embodiments, the poly-A tail is designed relative to the lengthof the overall polynucleotide or the length of a particular region ofthe polynucleotide. This design can be based on the length of a codingregion, the length of a particular feature or region or based on thelength of the ultimate product expressed from the polynucleotides.

In this context, the poly-A tail can be 10, 20, 30, 40, 50, 60, 70, 80,90, or 100% greater in length than the polynucleotide or featurethereof. The poly-A tail can also be designed as a fraction of thepolynucleotides to which it belongs. In this context, the poly-A tailcan be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the totallength of the construct, a construct region or the total length of theconstruct minus the poly-A tail. Further, engineered binding sites andconjugation of polynucleotides for Poly-A binding protein can enhanceexpression.

Additionally, multiple distinct polynucleotides can be linked togethervia the PABP (Poly-A binding protein) through the 3′-end using modifiednucleotides at the 3′-terminus of the poly-A tail. Transfectionexperiments can be conducted in relevant cell lines at and proteinproduction can be assayed by ELISA at 12 hr, 24 hr, 48 hr, 72 hr and day7 post-transfection.

In some embodiments, the polynucleotides of the present disclosure aredesigned to include a polyA-G Quartet region. The G-quartet is a cyclichydrogen bonded array of four guanine nucleotides that can be formed byG-rich sequences in both DNA and RNA. In this embodiment, the G-quartetis incorporated at the end of the poly-A tail. The resultantpolynucleotide is assayed for stability, protein production and otherparameters including half-life at various time points. It has beendiscovered that the polyA-G quartet results in protein production froman mRNA equivalent to at least 75% of that seen using a poly-A tail of120 nucleotides alone.

15. Start Codon Region

The present disclosure also includes a polynucleotide that comprisesboth a start codon region and the polynucleotide described herein (e.g.,a polynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide). In some embodiments, the polynucleotides of thepresent disclosure can have regions that are analogous to or functionlike a start codon region.

In some embodiments, the translation of a polynucleotide can initiate ona codon that is not the start codon AUG. Translation of thepolynucleotide can initiate on an alternative start codon such as, butnot limited to, ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU,TTG/UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 andMatsuda and Mauro PLoS ONE, 2010 5:11; the contents of each of which areherein incorporated by reference in its entirety).

As a non-limiting example, the translation of a polynucleotide begins onthe alternative start codon ACG. As another non-limiting example,polynucleotide translation begins on the alternative start codon CTG orCUG. As yet another non-limiting example, the translation of apolynucleotide begins on the alternative start codon GTG or GUG.

Nucleotides flanking a codon that initiates translation such as, but notlimited to, a start codon or an alternative start codon, are known toaffect the translation efficiency, the length and/or the structure ofthe polynucleotide. (See, e.g., Matsuda and Mauro PLoS ONE, 2010 5:11;the contents of which are herein incorporated by reference in itsentirety). Masking any of the nucleotides flanking a codon thatinitiates translation can be used to alter the position of translationinitiation, translation efficiency, length and/or structure of apolynucleotide.

In some embodiments, a masking agent can be used near the start codon oralternative start codon in order to mask or hide the codon to reduce theprobability of translation initiation at the masked start codon oralternative start codon. Non-limiting examples of masking agents includeantisense locked nucleic acids (LNA) polynucleotides and exon-junctioncomplexes (EJCs) (See, e.g., Matsuda and Mauro describing masking agentsLNA polynucleotides and EJCs (PLoS ONE, 2010 5:11); the contents ofwhich are herein incorporated by reference in its entirety).

In another embodiment, a masking agent can be used to mask a start codonof a polynucleotide in order to increase the likelihood that translationwill initiate on an alternative start codon. In some embodiments, amasking agent can be used to mask a first start codon or alternativestart codon in order to increase the chance that translation willinitiate on a start codon or alternative start codon downstream to themasked start codon or alternative start codon.

In some embodiments, a start codon or alternative start codon can belocated within a perfect complement for a miR binding site. The perfectcomplement of a miR binding site can help control the translation,length and/or structure of the polynucleotide similar to a maskingagent. As a non-limiting example, the start codon or alternative startcodon can be located in the middle of a perfect complement for a miRNAbinding site. The start codon or alternative start codon can be locatedafter the first nucleotide, second nucleotide, third nucleotide, fourthnucleotide, fifth nucleotide, sixth nucleotide, seventh nucleotide,eighth nucleotide, ninth nucleotide, tenth nucleotide, eleventhnucleotide, twelfth nucleotide, thirteenth nucleotide, fourteenthnucleotide, fifteenth nucleotide, sixteenth nucleotide, seventeenthnucleotide, eighteenth nucleotide, nineteenth nucleotide, twentiethnucleotide or twenty-first nucleotide.

In another embodiment, the start codon of a polynucleotide can beremoved from the polynucleotide sequence in order to have thetranslation of the polynucleotide begin on a codon that is not the startcodon. Translation of the polynucleotide can begin on the codonfollowing the removed start codon or on a downstream start codon or analternative start codon. In a non-limiting example, the start codon ATGor AUG is removed as the first 3 nucleotides of the polynucleotidesequence in order to have translation initiate on a downstream startcodon or alternative start codon. The polynucleotide sequence where thestart codon was removed can further comprise at least one masking agentfor the downstream start codon and/or alternative start codons in orderto control or attempt to control the initiation of translation, thelength of the polynucleotide and/or the structure of the polynucleotide.

16. Stop Codon Region

The present disclosure also includes a polynucleotide that comprisesboth a stop codon region and the polynucleotide described herein (e.g.,a polynucleotide comprising a nucleotide sequence encoding an antibody).In some embodiments, the polynucleotides of the present disclosure caninclude at least two stop codons before the 3′ untranslated region(UTR). The stop codon can be selected from TGA, TAA and TAG in the caseof DNA, or from UGA, UAA and UAG in the case of RNA. In someembodiments, the polynucleotides of the present disclosure include thestop codon TGA in the case or DNA, or the stop codon UGA in the case ofRNA, and one additional stop codon. In a further embodiment the additionstop codon can be TAA or UAA. In another embodiment, the polynucleotidesof the present disclosure include three consecutive stop codons, fourstop codons, or more.

17. Polynucleotide Comprising an mRNA Encoding an Antibody Polypeptide

In certain embodiments, a polynucleotide of the present disclosure, forexample a polynucleotide comprising an mRNA nucleotide sequence encodingan anti-CHIKV antibody polypeptide, comprises from 5′ to 3′ end:

-   -   (i) a 5′ cap provided above;    -   (ii) a 5′ UTR, such as a sequence provided above;    -   (iii) an open reading frame encoding an anti-CHIKV antibody        polypeptide, e.g., a sequence optimized nucleic acid sequence        encoding an anti-CHIKV antibody polypeptide disclosed herein;    -   (iv) at least one stop codon;    -   (v) a 3′ UTR, such as a sequence provided above; and    -   (vi) a poly-A tail provided above.

In some embodiments, the polynucleotide further comprises a miRNAbinding site, e.g., a miRNA binding site that binds to miRNA-142. Insome embodiments, the 5′UTR comprises the miRNA binding site. In someembodiments, the 3′ UTR comprises the miRNA binding site.

In some embodiments, a polynucleotide of the present disclosurecomprises a nucleotide sequence encoding a polypeptide sequence at least70%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100% identical to the protein sequence of an anti-CHIKV antibodypolypeptide described herein, such as a heavy chain polypeptide of ananti-CHIKV antibody (SEQ ID NO: 1) or a light chain polypeptide of ananti-CHIKV antibody (SEQ ID NO: 3).

In some embodiments, a polynucleotide of the present disclosurecomprises a nucleotide sequence comprising an open reading frame (ORF)that is at least 70%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to SEQ ID NO: 2 or SEQ ID NO:4.

In some embodiments, a polynucleotide of the present disclosurecomprises a nucleotide sequence comprising an open reading frame (ORF)that is at least 70%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to a nucleotide sequence encoding aheavy chain variable region of an anti-CHIKV antibody, e.g., nucleotides61-426 of SEQ ID NO:2.

In some embodiments, a polynucleotide of the present disclosurecomprises a nucleotide sequence comprising an open reading frame (ORF)that is at least 70%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to a nucleotide sequence encoding aheavy chain of an anti-CHIKV antibody, e.g., nucleotides 61-1416 of SEQID NO:2.

In some embodiments, a polynucleotide of the present disclosurecomprises a nucleotide sequence comprising an open reading frame (ORF)that is at least 70%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to a nucleotide sequence encoding alight chain variable region of an anti-CHIKV antibody, e.g., nucleotides61-384 of SEQ ID NO:4.

In some embodiments, a polynucleotide of the present disclosurecomprises a nucleotide sequence comprising an open reading frame (ORF)that is at least 70%, at least 80%, at least 81%, at least 82%, at least83%, at least 84%, at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% identical to a nucleotide sequence encoding alight chain of an anti-CHIKV antibody, e.g., nucleotides 61-705 of SEQID NO:4.

In some embodiments, a polynucleotide of the present disclosure, forexample a polynucleotide comprising an mRNA nucleotide sequence encodinga polypeptide, comprises (1) a 5′ cap provided above, for example, CAP1,(2) a 5′ UTR, (3) a nucleotide sequence ORF selected from SEQ ID NO:2 orSEQ ID NO:4, (3) a stop codon, (4) a 3′ UTR, and (5) a poly-A tailprovided above, for example, a poly-A tail of about 100 residues.

Exemplary anti-CHIKV antibody nucleotide constructs are described below:

SEQ ID NO:5 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO:13,anti-CHIKV antibody nucleotide ORF of SEQ ID NO:2, and 3′ UTR of SEQ IDNO:14.

SEQ ID NO:6 consists from 5′ to 3′ end: 5′ UTR of SEQ ID NO:13,anti-CHIKV antibody nucleotide ORF of SEQ ID NO:4, and 3′ UTR of SEQ IDNO:14.

In certain embodiments, in constructs with SEQ ID NOs. 5 and 6, alluracils therein are methylpseudouracils. In certain embodiments, inconstructs with SEQ ID NOs.: 5 and 6, all uracils therein are5-methoxyuracils.

In some embodiments, a polynucleotide of the present disclosure, forexample a polynucleotide comprising an mRNA nucleotide sequence encodingan anti-CHIKV antibody polypeptide, comprises (1) a 5′ cap providedabove, for example, CAP1, (2) a nucleotide sequence selected from SEQ IDNO:5 or SEQ ID NO:6, and (3) a poly-A tail provided above, for example,a poly A tail of ˜100 residues. In certain embodiments, in constructswith SEQ ID NOs.: 5 and 6, all uracils therein are N1methylpseudouracils. In certain embodiments, in constructs with SEQ IDNOs.: 5 and 6, all uracils therein are 5-methoxyuracils.

TABLE 5 Modified mRNA constructs including ORFs encoding a human anti-CHIKV antibody polypeptide (each of constructs #1 to #4 comprises a Cap15′ terminal cap and a 3′ terminal PolyA region) Anti-CHIKV 5′ UTR ORF 3′UTR antibody mRNA SEQ ID Name SEQ ID SEQ ID construct NO (Chemistry) NONO: #1 (SEQ ID NO: 5) 13 CHIKV24 heavy 2 14 chain (G5) #2 (SEQ ID NO: 6)13 CHIKV24 light 4 14 chain (G5)

18. Methods of Making Polynucleotides

The present disclosure also provides methods for making a polynucleotideof the present disclosure (e.g., a polynucleotide comprising anucleotide sequence encoding an anti-CHIKV antibody polypeptide) or acomplement thereof.

In some aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA) disclosedherein, and encoding an anti-CHIKV antibody polypeptide, can beconstructed using in vitro transcription. In other aspects, apolynucleotide (e.g., a RNA, e.g., an mRNA) disclosed herein, andencoding an antibody, can be constructed by chemical synthesis using anoligonucleotide synthesizer.

In other aspects, a polynucleotide (e.g., a RNA, e.g., an mRNA)disclosed herein, and encoding an anti-CHIKV antibody polypeptide ismade by using a host cell. In certain aspects, a polynucleotide (e.g., aRNA, e.g., an mRNA) disclosed herein, and encoding an anti-CHIKVantibody polypeptide is made by one or more combination of the IVT,chemical synthesis, host cell expression, or any other methods known inthe art.

Naturally occurring nucleosides, non-naturally occurring nucleosides, orcombinations thereof, can totally or partially naturally replaceoccurring nucleosides present in the candidate nucleotide sequence andcan be incorporated into a sequence-optimized nucleotide sequence (e.g.,a RNA, e.g., an mRNA) encoding an anti-CHIKV antibody polypeptide. Theresultant polynucleotides, e.g., mRNAs, can then be examined for theirability to produce protein and/or produce a therapeutic outcome.

a. In Vitro Transcription/Enzymatic Synthesis

The polynucleotides of the present disclosure disclosed herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) can be transcribed using an in vitro transcription(IVT) system. The system typically comprises a transcription buffer,nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.The NTPs can be selected from, but are not limited to, those describedherein including natural and unnatural (modified) NTPs. The polymerasecan be selected from, but is not limited to, T7 RNA polymerase, T3 RNApolymerase and mutant polymerases such as, but not limited to,polymerases able to incorporate polynucleotides disclosed herein. SeeU.S. Publ. No. US20130259923, which is herein incorporated by referencein its entirety.

Any number of RNA polymerases or variants can be used in the synthesisof the polynucleotides of the present disclosure. RNA polymerases can bemodified by inserting or deleting amino acids of the RNA polymerasesequence. As a non-limiting example, the RNA polymerase can be modifiedto exhibit an increased ability to incorporate a 2′-modified nucleotidetriphosphate compared to an unmodified RNA polymerase (see InternationalPublication WO2008078180 and U.S. Pat. No. 8,101,385; hereinincorporated by reference in their entireties).

Variants can be obtained by evolving an RNA polymerase, optimizing theRNA polymerase amino acid and/or nucleic acid sequence and/or by usingother methods known in the art. As a non-limiting example, T7 RNApolymerase variants can be evolved using the continuous directedevolution system set out by Esvelt et al. (Nature 472:499-503 (2011);herein incorporated by reference in its entirety) where clones of T7 RNApolymerase can encode at least one mutation such as, but not limited to,lysine at position 93 substituted for threonine (K93T), 14M, A7T, E63V,V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H,F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M2671,G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R,M401T, N410S, K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K,K577E, K577M, N601S, S684Y, L6991, K713E, N748D, Q754R, E775K, A827V,D851N or L864F. As another non-limiting example, T7 RNA polymerasevariants can encode at least mutation as described in U.S. Pub. Nos.20100120024 and 20070117112; herein incorporated by reference in theirentireties. Variants of RNA polymerase can also include, but are notlimited to, substitutional variants, conservative amino acidsubstitution, insertional variants, deletional variants and/or covalentderivatives.

In one aspect, the polynucleotide can be designed to be recognized bythe wild type or variant RNA polymerases. In doing so, thepolynucleotide can be modified to contain sites or regions of sequencechanges from the wild type or parent chimeric polynucleotide.

Polynucleotide or nucleic acid synthesis reactions can be carried out byenzymatic methods utilizing polymerases. Polymerases catalyze thecreation of phosphodiester bonds between nucleotides in a polynucleotideor nucleic acid chain. Currently known DNA polymerases can be dividedinto different families based on amino acid sequence comparison andcrystal structure analysis. DNA polymerase I (pol I) or A polymerasefamily, including the Klenow fragments of E. coli, Bacillus DNApolymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNAand DNA polymerases, is among the best studied of these families.Another large family is DNA polymerase a (pol u) or B polymerase family,including all eukaryotic replicating DNA polymerases and polymerasesfrom phages T4 and RB69. Although they employ similar catalyticmechanism, these families of polymerases differ in substratespecificity, substrate analog-incorporating efficiency, degree and ratefor primer extension, mode of DNA synthesis, exonuclease activity, andsensitivity against inhibitors.

DNA polymerases are also selected based on the optimum reactionconditions they require, such as reaction temperature, pH, and templateand primer concentrations. Sometimes a combination of more than one DNApolymerases is employed to achieve the desired DNA fragment size andsynthesis efficiency. For example, Cheng et al. increase pH, addglycerol and dimethyl sulfoxide, decrease denaturation times, increaseextension times, and utilize a secondary thermostable DNA polymerasethat possesses a 3′ to 5′ exonuclease activity to effectively amplifylong targets from cloned inserts and human genomic DNA. (Cheng et al.,PNAS 91:5695-5699 (1994), the contents of which are incorporated hereinby reference in their entirety). RNA polymerases from bacteriophage T3,T7, and SP6 have been widely used to prepare RNAs for biochemical andbiophysical studies. RNA polymerases, capping enzymes, and poly-Apolymerases are disclosed in the co-pending International PublicationNo. WO2014028429, the contents of which are incorporated herein byreference in their entirety.

In one aspect, the RNA polymerase which can be used in the synthesis ofthe polynucleotides of the present disclosure is a Syn5 RNA polymerase.(see Zhu et al. Nucleic Acids Research 2013, doi:10.1093/nar/gkt1193,which is herein incorporated by reference in its entirety). The Syn5 RNApolymerase was recently characterized from marine cyanophage Syn5 by Zhuet al. where they also identified the promoter sequence (see Zhu et al.Nucleic Acids Research 2013, the contents of which is hereinincorporated by reference in its entirety). Zhu et al. found that Syn5RNA polymerase catalyzed RNA synthesis over a wider range oftemperatures and salinity as compared to T7 RNA polymerase.Additionally, the requirement for the initiating nucleotide at thepromoter was found to be less stringent for Syn5 RNA polymerase ascompared to the T7 RNA polymerase making Syn5 RNA polymerase promisingfor RNA synthesis.

In one aspect, a Syn5 RNA polymerase can be used in the synthesis of thepolynucleotides described herein. As a non-limiting example, a Syn5 RNApolymerase can be used in the synthesis of the polynucleotide requiringa precise 3′-terminus.

In one aspect, a Syn5 promoter can be used in the synthesis of thepolynucleotides. As a non-limiting example, the Syn5 promoter can be5′-ATTGGGCACCCGTAAGGG-3′ (SEQ ID NO: 229), as described by Zhu et al.(Nucleic Acids Research 2013.

In one aspect, a Syn5 RNA polymerase can be used in the synthesis ofpolynucleotides comprising at least one chemical modification describedherein and/or known in the art (see e.g., the incorporation ofpseudo-UTP and 5Me-CTP described in Zhu et al. Nucleic Acids Research2013).

In one aspect, the polynucleotides described herein can be synthesizedusing a Syn5 RNA polymerase which has been purified using modified andimproved purification procedure described by Zhu et al. (Nucleic AcidsResearch 2013).

Various tools in genetic engineering are based on the enzymaticamplification of a target gene which acts as a template. For the studyof sequences of individual genes or specific regions of interest andother research needs, it is necessary to generate multiple copies of atarget gene from a small sample of polynucleotides or nucleic acids.Such methods can be applied in the manufacture of the polynucleotides ofthe present disclosure.

For example, polymerase chain reaction (PCR), strand displacementamplification (SDA), nucleic acid sequence-based amplification (NASBA),also called transcription mediated amplification (TMA), androlling-circle amplification (RCA) can be utilized in the manufacture ofone or more regions of the polynucleotides of the present disclosure.

Assembling polynucleotides or nucleic acids by a ligase is also widelyused. DNA or RNA ligases promote intermolecular ligation of the 5′ and3′ ends of polynucleotide chains through the formation of aphosphodiester bond.

b. Chemical Synthesis

Standard methods can be applied to synthesize an isolated polynucleotidesequence encoding an isolated polypeptide of interest, such as apolynucleotide of the present disclosure (e.g., a polynucleotidecomprising a nucleotide sequence encoding an antibody). For example, asingle DNA or RNA oligomer containing a codon-optimized nucleotidesequence coding for the particular isolated polypeptide can besynthesized. In other aspects, several small oligonucleotides coding forportions of the desired polypeptide can be synthesized and then ligated.In some aspects, the individual oligonucleotides typically contain 5′ or3′ overhangs for complementary assembly.

A polynucleotide disclosed herein (e.g., a RNA, e.g., an mRNA) can bechemically synthesized using chemical synthesis methods and potentialnucleobase substitutions known in the art. See, for example,International Publication Nos. WO2014093924, WO2013052523; WO2013039857,WO2012135805, WO2013151671; U.S. Publ. No. US20130115272; or U.S. Pat.Nos. U.S. Pat. No. 8,999,380 or 8,710,200, all of which are hereinincorporated by reference in their entireties.

c. Purification of Polynucleotides Encoding an Antibody

Purification of the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) can include, but is not limited to, polynucleotideclean-up, quality assurance and quality control. Clean-up can beperformed by methods known in the arts such as, but not limited to,AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads,LNA™ oligo-T capture probes (EXIQON® Inc., Vedbaek, Denmark) or HPLCbased purification methods such as, but not limited to, strong anionexchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC),and hydrophobic interaction HPLC (HIC-HPLC).

The term “purified” when used in relation to a polynucleotide such as a“purified polynucleotide” refers to one that is separated from at leastone contaminant. As used herein, a “contaminant” is any substance thatmakes another unfit, impure or inferior. Thus, a purified polynucleotide(e.g., DNA and RNA) is present in a form or setting different from thatin which it is found in nature, or a form or setting different from thatwhich existed prior to subjecting it to a treatment or purificationmethod.

In some embodiments, purification of a polynucleotide of the presentdisclosure (e.g., a polynucleotide comprising a nucleotide sequenceencoding an antibody) removes impurities that can reduce or remove anunwanted immune response, e.g., reducing cytokine activity.

In some embodiments, the polynucleotide of the present disclosure (e.g.,a polynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) is purified prior to administration using columnchromatography (e.g., strong anion exchange HPLC, weak anion exchangeHPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC(HIC-HPLC), or (LCMS)).

In some embodiments, the polynucleotide of the present disclosure (e.g.,a polynucleotide comprising a nucleotide sequence an anti-CHIKV antibodypolypeptide) purified using column chromatography (e.g., strong anionexchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC,hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)) presents increasedexpression of the encoded antibody compared to the expression levelobtained with the same polynucleotide of the present disclosure purifiedby a different purification method.

In some embodiments, a column chromatography (e.g., strong anionexchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC),hydrophobic interaction HPLC (HIC-HPLC), or (LCMS)) purifiedpolynucleotide comprises a nucleotide sequence encoding an anti-CHIKVantibody polypeptide comprising one or more of the point mutations knownin the art.

In some embodiments, the use of RP-HPLC purified polynucleotideincreases anti-CHIKV antibody polypeptide expression levels in cellswhen introduced into those cells, e.g., by 10-100%, i.e., at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about90%, at least about 95%, or at least about 100% with respect to theexpression levels of antibody in the cells before the RP-HPLC purifiedpolynucleotide was introduced in the cells, or after a non-RP-HPLCpurified polynucleotide was introduced in the cells.

In some embodiments, the use of RP-HPLC purified polynucleotideincreases functional anti-CHIKV antibody polypeptide expression levelsin cells when introduced into those cells, e.g., by 10-100%, i.e., atleast about 10%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, at least about 75%, at least about 80%, atleast about 90%, at least about 95%, or at least about 100% with respectto the functional expression levels of antibody in the cells before theRP-HPLC purified polynucleotide was introduced in the cells, or after anon-RP-HPLC purified polynucleotide was introduced in the cells.

In some embodiments, the use of RP-HPLC purified polynucleotideincreases detectable anti-CHIKV antibody polypeptide activity in cellswhen introduced into those cells, e.g., by 10-100%, i.e., at least about10%, at least about 20%, at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about90%, at least about 95%, or at least about 100% with respect to theactivity levels of functional antibody in the cells before the RP-HPLCpurified polynucleotide was introduced in the cells, or after anon-RP-HPLC purified polynucleotide was introduced in the cells.

In some embodiments, the purified polynucleotide is at least about 80%pure, at least about 85% pure, at least about 90% pure, at least about95% pure, at least about 96% pure, at least about 97% pure, at leastabout 98% pure, at least about 99% pure, or about 100% pure.

A quality assurance and/or quality control check can be conducted usingmethods such as, but not limited to, gel electrophoresis, UV absorbance,or analytical HPLC. In another embodiment, the polynucleotide can besequenced by methods including, but not limited toreverse-transcriptase-PCR.

d. Quantification of Expressed Polynucleotides Encoding Antibody

In some embodiments, the polynucleotides of the present disclosure(e.g., a polynucleotide comprising a nucleotide sequence encoding ananti-CHIKV antibody polypeptide), their expression products, as well asdegradation products and metabolites can be quantified according tomethods known in the art.

In some embodiments, the polynucleotides of the present disclosure canbe quantified in exosomes or when derived from one or more bodily fluid.As used herein “bodily fluids” include peripheral blood, serum, plasma,ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow,synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk,broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid orpre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid,pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle,bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions,mucosal secretion, stool water, pancreatic juice, lavage fluids fromsinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, andumbilical cord blood. Alternatively, exosomes can be retrieved from anorgan selected from the group consisting of lung, heart, pancreas,stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast,prostate, brain, esophagus, liver, and placenta.

In the exosome quantification method, a sample of not more than 2 mL isobtained from the subject and the exosomes isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.In the analysis, the level or concentration of a polynucleotide can bean expression level, presence, absence, truncation or alteration of theadministered construct. It is advantageous to correlate the level withone or more clinical phenotypes or with an assay for a human diseasebiomarker.

The assay can be performed using construct specific probes, cytometry,qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, massspectrometry, or combinations thereof while the exosomes can be isolatedusing immunohistochemical methods such as enzyme linked immunosorbentassay (ELISA) methods. Exosomes can also be isolated by size exclusionchromatography, density gradient centrifugation, differentialcentrifugation, nanomembrane ultrafiltration, immunoabsorbent capture,affinity purification, microfluidic separation, or combinations thereof.

These methods afford the investigator the ability to monitor, in realtime, the level of polynucleotides remaining or delivered. This ispossible because the polynucleotides of the present disclosure differfrom the endogenous forms due to the structural or chemicalmodifications.

In some embodiments, the polynucleotide can be quantified using methodssuch as, but not limited to, ultraviolet visible spectroscopy (UV/Vis).A non-limiting example of a UV/Vis spectrometer is a NANODROP®spectrometer (ThermoFisher, Waltham, MA). The quantified polynucleotidecan be analyzed in order to determine if the polynucleotide can be ofproper size, check that no degradation of the polynucleotide hasoccurred. Degradation of the polynucleotide can be checked by methodssuch as, but not limited to, agarose gel electrophoresis, HPLC basedpurification methods such as, but not limited to, strong anion exchangeHPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), andhydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-massspectrometry (LCMS), capillary electrophoresis (CE) and capillary gelelectrophoresis (CGE).

19. Pharmaceutical Compositions and Formulations Provided herein arecompositions (e.g., pharmaceutical compositions), methods, kits andreagents for prevention and/or treatment of disease in humans and othermammals. The present invention provides pharmaceutical compositions andformulations that comprise any of the polynucleotides described above.In some embodiments, the composition or formulation further comprises adelivery agent.

In some embodiments, the composition or formulation can contain apolynucleotide comprising a sequence optimized nucleic acid sequencedisclosed herein which encodes an anti-CHIKV antibody polypeptide. Insome embodiments, the composition or formulation can contain apolynucleotide (e.g., a RNA, e.g., an mRNA) comprising a polynucleotide(e.g., an ORF) having significant sequence identity to a sequenceoptimized nucleic acid sequence disclosed herein which encodes ananti-CHIKV antibody polypeptide. In some embodiments, the polynucleotidefurther comprises a miRNA binding site, e.g., a miRNA binding site thatbinds miR-126, miR-142, miR-144, miR-146, miR-150, miR-155, miR-16,miR-21, miR-223, miR-24, miR-27 and miR-26a.

In some embodiments, the pharmaceutical compositions described hereinhave a first polynucleotide comprising a first mRNA comprising (i) afirst 5′ UTR, (ii) a first open reading frame (ORF) encoding a firstpolypeptide comprising a heavy chain antibody sequence of SEQ ID NO:1,wherein the first ORF comprises a first nucleic acid sequence that is atleast 80% identical to SEQ ID NO:2, (iii) a first stop codon, and (iv) afirst 3′ UTR; a second polynucleotide comprising a second mRNAcomprising (i) a second 5′ UTR, (ii) a second ORF encoding a secondpolypeptide comprising the light chain antibody sequence of SEQ ID NO:3,wherein the second ORF comprises a second nucleic acid sequence that isat least 80% identical to SEQ ID NO:4, (iii) a second stop codon, and(iv) a second 3′ UTR; and a delivery agent, wherein the firstpolypeptide when paired with the second polypeptide forms ananti-Chikungunya virus antibody.

In some embodiments, the pharmaceutical compositions described hereinhave a first polynucleotide comprising a first mRNA comprising (i) afirst 5′ UTR, (ii) a first open reading frame (ORF) encoding a firstpolypeptide comprising the heavy chain variable region of the heavychain antibody sequence of SEQ ID NO:1, wherein the first ORF comprisesa first nucleic acid sequence that is at least 80% identical tonucleotides 61-426 of SEQ ID NO:2, (iii) a first stop codon, and (iv) afirst 3′ UTR; a second polynucleotide comprising a second mRNAcomprising (i) a second 5′ UTR, (ii) a second ORF encoding a secondpolypeptide comprising the light chain variable region of the lightchain antibody sequence of SEQ ID NO:3, wherein the second ORF comprisesa second nucleic acid sequence that is at least 80% identical tonucleotides 61-384 of SEQ ID NO:4, (iii) a second stop codon, and (iv) asecond 3′ UTR; and a delivery agent, wherein the first polypeptide whenpaired with the second polypeptide forms an anti-Chikungunya virusantibody or an anti-Chikungunya virus antibody fragment.

In some embodiments, the pharmaceutical compositions described hereinhave a first polynucleotide comprising a first mRNA comprising (i) afirst 5′ UTR, (ii) a first open reading frame (ORF) encoding a firstpolypeptide comprising the heavy chain of the heavy chain antibodysequence of SEQ ID NO:1, wherein the first ORF comprises a first nucleicacid sequence that is at least 80% identical to nucleotides 61-1416 ofSEQ ID NO:2, (iii) a first stop codon, and (iv) a first 3′ UTR; a secondpolynucleotide comprising a second mRNA comprising (i) a second 5′ UTR,(ii) a second ORF encoding a second polypeptide comprising the lightchain of the light chain antibody sequence of SEQ ID NO:3, wherein thesecond ORF comprises a second nucleic acid sequence that is at least 80%identical to nucleotides 61-705 of SEQ ID NO:4, (iii) a second stopcodon, and (iv) a second 3′ UTR; and a delivery agent, wherein the firstpolypeptide when paired with the second polypeptide forms ananti-Chikungunya virus antibody or an anti-Chikungunya virus antibodyfragment.

In some embodiments, a pharmaceutical composition described herein has afirst mRNA with the nucleic acid sequence of SEQ ID NO:5 and a secondmRNA with the nucleic acid sequence of SEQ ID NO:6.

In some embodiments, a first mRNA comprising a first open reading frame(ORF) encoding a first polypeptide comprising a heavy chain variableregion of an anti-CHIKV antibody (e.g., an anti-CHIKV heavy chainpolypeptide) is co-formulated in a lipid nanoparticle (LNP) with asecond mRNA comprising a second open reading frame (ORF) encoding asecond polypeptide comprising a light chain variable region of ananti-CHIKV (e.g., an anti-CHIKV light chain polypeptide) at a ratio w/wof 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 3:2, 5:7, or 7:9, respectively. In someembodiments, a first mRNA comprising a first open reading frame (ORF)encoding a first polypeptide comprising a heavy chain variable region ofan anti-CHIKV antibody (e.g., an anti-CHIKV heavy chain polypeptide) anda second mRNA comprising a first open reading frame (ORF) encoding asecond polypeptide comprising a light chain variable region of ananti-CHIKV (e.g., an anti-CHIKV light chain polypeptide) areco-formulated at a 2:1 ratio w/w (2:1 first mRNA:second mRNA) in anlipid nanoparticle (LNP). In some embodiments, the pharmaceuticalcomposition comprises an LNP with a first mRNA with the nucleic acidsequence of SEQ ID NO:5 and a second mRNA with the nucleic acid sequenceof SEQ ID NO:6 at a ratio of 2:1 w/w.

In some embodiments, a pharmaceutical composition has a first mRNAcomprising a first open reading frame (ORF) encoding a first polypeptidecomprising a heavy chain variable region of an anti-chikungunya virusantibody and a second mRNA comprising a second ORF encoding a secondpolypeptide comprising a light chain variable region of theanti-chikungunya virus antibody, wherein the first polypeptide and thesecond polypeptide pair to form the anti-chikungunya virus antibody, andwherein the pharmaceutical composition when administered to a humansubject in need thereof as a single dose administration is sufficientto: (i) protect the human subject from chikungunya virus infection,after exposure to a chikungunya virus, for at least 24 hours, 48 hours,72 hours, 96 hours, 168 hours, 336 hours, or 720 hours after the singledose administration; (ii) protect the human subject from onset ofchikungunya fever, after exposure to a chikungunya virus, for at least24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 336 hours, or 720hours after the single dose administration; and/or (iii) providesystemic production of the anti-chikungunya virus antibody in the humansubject at a level of at least 5 μg/ml, 10 μg/ml, 15 μg/ml, 20 μg/ml, 25μg/ml, or 30 μg/ml for at least 24 hours, 48 hours, 72 hours, 96 hours,168 hours, 336 hours, or 720 hours after the single dose administration.

Pharmaceutical compositions or formulation can optionally comprise oneor more additional active substances, e.g., therapeutically and/orprophylactically active substances. Pharmaceutical compositions orformulation of the present invention can be sterile and/or pyrogen-free.General considerations in the formulation and/or manufacture ofpharmaceutical agents can be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety). Insome embodiments, compositions are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to polynucleotides to bedelivered as described herein.

Formulations and pharmaceutical compositions described herein can beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofassociating the active ingredient with an excipient and/or one or moreother accessory ingredients, and then, if necessary and/or desirable,dividing, shaping and/or packaging the product into a desired single- ormulti-dose unit.

A pharmaceutical composition or formulation in accordance with thepresent disclosure can be prepared, packaged, and/or sold in bulk, as asingle unit dose, and/or as a plurality of single unit doses. As usedherein, a “unit dose” refers to a discrete amount of the pharmaceuticalcomposition comprising a predetermined amount of the active ingredient.The amount of the active ingredient is generally equal to the dosage ofthe active ingredient that would be administered to a subject and/or aconvenient fraction of such a dosage such as, for example, one-half orone-third of such a dosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the present disclosure canvary, depending upon the identity, size, and/or condition of the subjectbeing treated and further depending upon the route by which thecomposition is to be administered.

In some embodiments, the compositions and formulations described hereincan contain at least one polynucleotide of the invention. As anon-limiting example, the composition or formulation can contain 1, 2,3, 4 or 5 polynucleotides of the invention. In some embodiments, thecompositions or formulations described herein can comprise more than onetype of polynucleotide. In some embodiments, the composition orformulation can comprise a polynucleotide in linear and circular form.In another embodiment, the composition or formulation can comprise acircular polynucleotide and an in vitro transcribed (IVT)polynucleotide. In yet another embodiment, the composition orformulation can comprise an IVT polynucleotide, a chimericpolynucleotide and a circular polynucleotide.

Although the descriptions of pharmaceutical compositions andformulations provided herein are principally directed to pharmaceuticalcompositions and formulations that are suitable for administration tohumans, it will be understood by the skilled artisan that suchcompositions are generally suitable for administration to any otheranimal, e.g., to non-human animals, e.g. non-human mammals.

The present invention provides pharmaceutical formulations that comprisea polynucleotide described herein (e.g., a polynucleotide comprising anucleotide sequence encoding an anti-CHIKV antibody polypeptide). Thepolynucleotides described herein can be formulated using one or moreexcipients to: (1) increase stability; (2) increase cell transfection;(3) permit the sustained or delayed release (e.g., from a depotformulation of the polynucleotide); (4) alter the biodistribution (e.g.,target the polynucleotide to specific tissues or cell types); (5)increase the translation of encoded protein in vivo; and/or (6) alterthe release profile of encoded protein in vivo. In some embodiments, thepharmaceutical formulation further comprises a delivery agentcomprising, e.g., a compound having the Formula (I), e.g., any ofCompounds 1-232, e.g., Compound II; a compound having the Formula (III),(IV), (V), or (VI), e.g., any of Compounds 233-342, e.g., Compound VI;or a compound having the Formula (VIII), e.g., any of Compounds 419-428,e.g., Compound I, or any combination thereof. In some embodiments, thedelivery agent comprises Compound II, DSPC, Cholesterol, and Compound Ior PEG-DMG, e.g., with a mole ratio of about 50:10:38.5:1.5. In someembodiments, the delivery agent comprises Compound II, DSPC,Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio of about47.5:10.5:39.0:3.0. In some embodiments, the delivery agent comprisesCompound II, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with amole ratio of about 50:10:38:2. In some embodiments, the delivery agentcomprises Compound VI, DSPC, Cholesterol, and Compound I or PEG-DMG,e.g., with a mole ratio of about 50:10:38.5:1.5. In some embodiments,the delivery agent comprises Compound VI, DSPC, Cholesterol, andCompound I or PEG-DMG, e.g., with a mole ratio of about47.5:10.5:39.0:3.0.

A pharmaceutically acceptable excipient, as used herein, includes, butare not limited to, any and all solvents, dispersion media, or otherliquid vehicles, dispersion or suspension aids, diluents, granulatingand/or dispersing agents, surface active agents, isotonic agents,thickening or emulsifying agents, preservatives, binders, lubricants oroil, coloring, sweetening or flavoring agents, stabilizers,antioxidants, antimicrobial or antifungal agents, osmolality adjustingagents, pH adjusting agents, buffers, chelants, cyoprotectants, and/orbulking agents, as suited to the particular dosage form desired. Variousexcipients for formulating pharmaceutical compositions and techniquesfor preparing the composition are known in the art (see Remington: TheScience and Practice of Pharmacy, 21st Edition, A. R. Gennaro(Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporatedherein by reference in its entirety).

Exemplary diluents include, but are not limited to, calcium or sodiumcarbonate, calcium phosphate, calcium hydrogen phosphate, sodiumphosphate, lactose, sucrose, cellulose, microcrystalline cellulose,kaolin, mannitol, sorbitol, etc., and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are notlimited to, starches, pregelatinized starches, or microcrystallinestarch, alginic acid, guar gum, agar, poly(vinyl-pyrrolidone),(providone), cross-linked poly(vinyl-pyrrolidone) (crospovidone),cellulose, methylcellulose, carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), magnesium aluminum silicate(VEEGUM®), sodium lauryl sulfate, etc., and/or combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g., acacia, agar, alginic acid,sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin,gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin),sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate[TWEEN®80], sorbitan monopalmitate [SPAN®40], glyceryl monooleate,polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g.,CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether[BRIJ®30]), PLUORINC®F 68, POLOXAMER®188, etc. and/or combinationsthereof.

Exemplary binding agents include, but are not limited to, starch,gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses,lactose, lactitol, mannitol), amino acids (e.g., glycine), natural andsynthetic gums (e.g., acacia, sodium alginate), ethylcellulose,hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., andcombinations thereof.

Oxidation is a potential degradation pathway for mRNA, especially forliquid mRNA formulations. In order to prevent oxidation, antioxidantscan be added to the formulations. Exemplary antioxidants include, butare not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate,benzyl alcohol, butylated hydroxyanisole, m-cresol, methionine,butylated hydroxytoluene, monothioglycerol, sodium or potassiummetabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc.,and combinations thereof.

Exemplary chelating agents include, but are not limited to,ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, fumaric acid, malic acid, phosphoric acid, sodiumedetate, tartaric acid, trisodium edetate, etc., and combinationsthereof.

Exemplary antimicrobial or antifungal agents include, but are notlimited to, benzalkonium chloride, benzethonium chloride, methylparaben, ethyl paraben, propyl paraben, butyl paraben, benzoic acid,hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodiumsorbate, sodium propionate, sorbic acid, etc., and combinations thereof.

Exemplary preservatives include, but are not limited to, vitamin A,vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid,butylated hydroxyanisol, ethylenediamine, sodium lauryl sulfate (SLS),sodium lauryl ether sulfate (SLES), etc., and combinations thereof.

In some embodiments, the pH of polynucleotide solutions are maintainedbetween pH and pH 8 to improve stability. Exemplary buffers to controlpH can include, but are not limited to sodium phosphate, sodium citrate,sodium succinate, histidine (or histidine-HCl), sodium malate, sodiumcarbonate, etc., and/or combinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt,hydrogenated vegetable oils, polyethylene glycol, sodium benzoate,sodium or magnesium lauryl sulfate, etc., and combinations thereof.

The pharmaceutical composition or formulation described here can containa cryoprotectant to stabilize a polynucleotide described herein duringfreezing. Exemplary cryoprotectants include, but are not limited tomannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., andcombinations thereof.

The pharmaceutical composition or formulation described here can containa bulking agent in lyophilized polynucleotide formulations to yield a“pharmaceutically elegant” cake, stabilize the lyophilizedpolynucleotides during long term (e.g., 36 month) storage. Exemplarybulking agents of the present invention can include, but are not limitedto sucrose, trehalose, mannitol, glycine, lactose, raffinose, andcombinations thereof.

In some embodiments, the pharmaceutical composition or formulationfurther comprises a delivery agent. The delivery agent of the presentdisclosure can include, without limitation, liposomes, lipidnanoparticles, lipidoids, polymers, lipoplexes, microvesicles, exosomes,peptides, proteins, cells transfected with polynucleotides,hyaluronidase, nanoparticle mimics, nanotubes, conjugates, andcombinations thereof.

The polynucleotides encoding anti-CHIKV antibodies can be used astherapeutic or prophylactic agents. Pharmaceutical compositions can beadministered once, twice, three times, four times or more. In someaspects, the compositions can be administered to an infected individualto achieve a therapeutic response. Dosing may need to be adjustedaccordingly.

It is envisioned that there may be situations where persons are at riskfor infection with more than one strain of type of infectious agent. RNA(mRNA) therapeutic treatments are particularly amenable to combinationvaccination approaches due to a number of factors including, but notlimited to, speed of manufacture, ability to rapidly tailor treatmentsto accommodate perceived geographical threat, and the like. To protectagainst more than one strain of virus, a combination treatment can beadministered that includes mRNA encoding at least one polypeptide (orportion thereof) that binds to an antigen of a first strain, and furtherincludes mRNA encoding at least one polypeptide (or portion thereof)that binds to an antigen of a second strain of virus. RNAs (mRNAs) canbe co-formulated, for example, in a single lipid nanoparticle (LNP) orcan be formulated in separate LNPs destined for co-administration.

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments, the therapeutically effective dose is a dose listedin a package insert for the treatment. A prophylactic therapy as usedherein refers to a therapy that prevents, to some extent, the infectionfrom increasing. The infection may be prevented completely or partially.

The methods of the mention involve, in some aspects, passivelyimmunizing a mammalian subject against a chikungunya virus infection.The method involves administering to the subject a compositioncomprising at least one RNA polynucleotide having an open reading frameencoding at least one antibody polypeptide (e.g., the heavy and lightchains of an antibody) that targets (e.g., binds to) a chikungunya virusprotein. In some aspects, methods of the present disclosure provideprophylactic treatments against a chikungunya virus infection.

Therapeutic methods of treatment are also included within the invention.Methods of treating a chikungunya virus infection in a subject areprovided in aspects of the disclosure. The method involves administeringto the subject having a chikungunya virus infection a compositioncomprising at least one RNA polynucleotide having an open reading frameencoding at least one anti-CHIKV antibody polypeptide that targets(e.g., binds to) a chikungunya virus protein.

As used herein, the terms “treat”, “treated”, or “treating” when usedwith respect to a disorder such as a viral infection, refers to atreatment which increases the resistance of a subject to development ofthe disease or, in other words, decreases the likelihood that thesubject will develop the disease in response to infection with the virusas well as a treatment after the subject has developed the disease inorder to fight the infection or prevent the infection from becomingworse.

An “effective amount” of an mRNA therapeutic is provided based, at leastin part, on the target tissue, target cell type, means ofadministration, physical characteristics of the polynucleotide (e.g.,size, and extent of modified nucleosides), and other components of theRNA treatment, and other determinants. Increased antibody production maybe demonstrated by increased cell transfection (the percentage of cellstransfected with the RNA treatment), increased protein translation fromthe polynucleotide, decreased nucleic acid degradation (as demonstrated,for example, by increased duration of protein translation from amodified polynucleotide), or altered response of the host cell.

In some embodiments, the polynucleotides described herein in accordancewith the present disclosure may be used for treatment of the disease.

The polynucleotides (e.g., mRNA) described herein may be administeredprophylactically or therapeutically as part of an active immunizationscheme to healthy individuals or early in infection during theincubation phase or during active infection after onset of symptoms. Insome embodiments, the amount of polynucleotides of the presentdisclosure provided to a cell, a tissue or a subject may be an amounteffective for immune prophylaxis.

In some embodiments, the polynucleotides (e.g., mRNA) described hereincan be used in combination with another therapy or treatment forchikungunya infection. By way of example, one or more polynucleotidesdescribed herein can be administered to a subject with a chikungunyavirus infection in combination with, e.g., supportive care e.g., rest,fluids, et.), antipyretics, and/or analgesics.

The polynucleotides (e.g., mRNA) described herein may be administeredwith other prophylactic or therapeutic compounds. As a non-limitingexample, a prophylactic or therapeutic compound may be a vaccinecontaining a virus treatment with or without an adjuvant or a booster.As used herein, when referring to a prophylactic composition, such as atreatment or vaccine, the term “booster” refers to an extraadministration of the prophylactic composition. A booster (or boostervaccine) may be given after an earlier administration of theprophylactic composition. The time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years,7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25years, 30 years, years, 40 years, 45 years, 50 years, 55 years, 60years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95years, or more than 99 years. In exemplary embodiments, the time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 week, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, or 1 year.

In some embodiments, the polynucleotides (e.g., mRNA) described hereinmay be administered subcutaneously, intraocularly, intravitreally,parenterally, subcutaneously, intravenously, intracerebro-ventricularly,intramuscularly, intrathecally, orally, intraperitoneally, by oral ornasal inhalation, or by direct injection to one or more cells, tissues,or organs.

Provided herein are pharmaceutical compositions including thepolynucleotides described herein (e.g., mRNA) and/or complexesoptionally in combination with one or more pharmaceutically acceptableexcipients.

The polynucleotides described herein (e.g., mRNA) may be formulated oradministered in combination with one or more pharmaceutically-acceptableexcipients. In some embodiments, compositions comprise at least oneadditional active substances, such as, for example, atherapeutically-active substance, a prophylactically-active substance,or a combination of both. Treatment compositions may be sterile,pyrogen-free or both sterile and pyrogen-free. General considerations inthe formulation and/or manufacture of pharmaceutical agents, such astreatment compositions, may be found, for example, in Remington: TheScience and Practice of Pharmacy 21st ed., Lippincott Williams &Wilkins, 2005 (incorporated herein by reference in its entirety).

In some embodiments, RNA treatments (e.g., a composition containing atleast one mRNA described herein) are administered to humans, humanpatients, or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to the polynucleotide orpolynucleotides contained in a therapeutic composition therein, forexample, RNA polynucleotides (e.g., mRNA polynucleotides) encodinganti-CHIKV antibody polypeptides.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

In some embodiments, the RNA treatment may include one or morestabilizing elements. Stabilizing elements may include, for instance, ahistone stem-loop. A stem-loop binding protein (SLBP), a 32 kDa proteinhas been identified. It is associated with the histone stem-loop at the3′-end of the histone messages in both the nucleus and the cytoplasm.Its expression level is regulated by the cell cycle; it is peaks duringthe S-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, the RNA treatments include a coding region, atleast one histone stem-loop, and optionally, a poly(A) sequence orpolyadenylation signal. The poly(A) sequence or polyadenylation signalgenerally should enhance the expression level of the encoded protein.The encoded protein, in some embodiments, is not a histone protein, areporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, EGFP), ora marker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:guanine phosphoribosyl transferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. It has been found that the synergistic effectof the combination of poly(A) and at least one histone stem-loop doesnot depend on the order of the elements or the length of the poly(A)sequence.

20. Delivery Agents

In one set of embodiments, lipid nanoparticles (LNPs) are provided. Inone embodiment, a lipid nanoparticle comprises lipids including anionizable lipid, a structural lipid, a phospholipid, and mRNA. Each ofthe LNPs described herein may be used as a formulation for the mRNAdescribed herein. In one embodiment, a lipid nanoparticle comprises anionizable lipid, a structural lipid, a phospholipid, and mRNA. In someembodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid,a phospholipid and a structural lipid. In some embodiments, the LNP hasa molar ratio of about 20-60% ionizable lipid: about 5-25% phospholipid:about 25-55% structural lipid; and about 0.5-15% PEG-modified lipid. Insome embodiments, the LNP comprises a molar ratio of about 50% ionizablelipid, about 1.5% PEG-modified lipid, about 38.5% structural lipid andabout 10% phospholipid. In some embodiments, the LNP comprises a molarratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5%structural lipid and about 10% phospholipid. In some embodiments, theLNP comprises a molar ratio of about 50% ionizable lipid, about 2% PEGlipid, about 38% structural lipid and about 10% phospholipid. In someembodiments, the ionizable lipid is an ionizable amino or cationic lipidand the phospholipid is a neutral lipid, and the structural lipid is acholesterol. In some embodiments, the LNP has a molar ratio of50:38.5:10:1.5 of ionizable lipid: cholesterol:DSPC: PEG2000-DMG. Insome embodiments, the LNP has a molar ratio of 50:38:10:2 of ionizablelipid: cholesterol:DSPC: PEG-lipid.

a. Lipid Compound

The present disclosure provides pharmaceutical compositions withadvantageous properties. The lipid compositions described herein may beadvantageously used in lipid nanoparticle compositions for the deliveryof therapeutic and/or prophylactic agents, e.g., mRNAs, to mammaliancells or organs. For example, the lipids described herein have little orno immunogenicity. For example, the lipid compounds disclosed hereinhave a lower immunogenicity as compared to a reference lipid (e.g., MC3,KC2, or DLinDMA). For example, a formulation comprising a lipiddisclosed herein and a therapeutic or prophylactic agent, e.g., mRNA,has an increased therapeutic index as compared to a correspondingformulation which comprises a reference lipid (e.g., MC3, KC2, orDLinDMA) and the same therapeutic or prophylactic agent.

In certain embodiments, the present application provides pharmaceuticalcompositions comprising:

-   -   (a) a polynucleotide comprising a nucleotide sequence encoding        an anti-CHIKV antibody polypeptide; and    -   (b) a delivery agent.        Lipid Nanoparticle Formulations

In some embodiments, nucleic acids of the invention (e.g. anti-CHIKVantibody mRNA) are formulated in a lipid nanoparticle (LNP). Lipidnanoparticles typically comprise ionizable cationic lipid, non-cationiclipid, sterol and PEG lipid components along with the nucleic acid cargoof interest. The lipid nanoparticles of the invention can be generatedusing components, compositions, and methods as are generally known inthe art, see for example PCT/US2016/052352; PCT/US2016/068300;PCT/US2017/037551; PCT/US2015/027400; PCT/US2016/047406;PCT/US2016000129; PCT/US2016/014280; PCT/US2016/014280;PCT/US2017/038426; PCT/US2014/027077; PCT/US2014/055394;PCT/US2016/52117; PCT/US2012/069610; PCT/US2017/027492;PCT/US2016/059575 and PCT/US2016/069491 all of which are incorporated byreference herein in their entirety.

Nucleic acids of the present disclosure (e.g. anti-CHIKV antibody mRNA)are typically formulated in lipid nanoparticle. In some embodiments, thelipid nanoparticle comprises at least one ionizable cationic lipid, atleast one non-cationic lipid, at least one sterol, and/or at least onepolyethylene glycol (PEG)-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of20-60% ionizable cationic lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 20-50%, 20-40%, 20-30%, 30-60%, 30-50%,30-40%, 40-60%, 40-50%, or 50-60% ionizable cationic lipid. In someembodiments, the lipid nanoparticle comprises a molar ratio of 20%, 30%,40%, 50, or 60% ionizable cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of5-25% non-cationic lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 5-20%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%,15-25%, 15-20%, or 20-25% non-cationic lipid. In some embodiments, thelipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, or 25%non-cationic lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of25-55% sterol. For example, the lipid nanoparticle may comprise a molarratio of 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30-50%, 30-45%,30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%,45-55%, 45-50%, or 50-55% sterol. In some embodiments, the lipidnanoparticle comprises a molar ratio of 25%, 30%, 35%, 38%, 40%, 45%,50%, or 55% sterol.

In some embodiments, the lipid nanoparticle comprises a molar ratio of0.5-15% PEG-modified lipid. For example, the lipid nanoparticle maycomprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%,2-10%, 2-5%, 5-15%, 5-10%, or 10-15%. In some embodiments, the lipidnanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG-modified lipid.

In some embodiments, the lipid nanoparticle comprises a molar ratio of20-60% ionizable cationic lipid, 5-25% non-cationic lipid, 25-55%sterol, and 0.5-15% PEG-modified lipid.

Ionizable Lipids

In some aspects, the ionizable lipids of the present disclosure may beone or more of compounds of Formula (I):

-   -   or their N-oxides, or salts or isomers thereof, wherein:    -   R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀        alkenyl, —R*YR″, —YR″, and —R″M′R′;    -   R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or        R₂ and R₃, together with the atom to which they are attached,        form a heterocycle or carbocycle;    -   R₄ is selected from the group consisting of hydrogen, a C₃₋₆        carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,        -   —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is            selected from a carbocycle, heterocycle, —OR,            —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,            —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,            —N(R)C(S)N(R)₂, —N(R)R₈,    -   —N(R)S(O)₂R₈, —O(C_(H)2)_(n)OR, —N(R)C(═NR₉)N(R)₂,        —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂,    -   —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,        —N(OR)C(O)N(R)₂,    -   —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂,        —C(═NR₉)N(R)₂,    -   —C(═NR₉)R, —C(O)N(R)OR, and —C(R)N(R)₂C(O)OR, and each n is        independently selected from 1, 2, 3, 4, and 5;    -   each R₅ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R₆ is independently selected from the group consisting of        C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   M and M′ are independently selected    -   from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—,        -   —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—,            —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a            heteroaryl group, in which M″ is a bond, C₁₋₁₃ alkyl or            C₂₋₁₃ alkenyl;        -   R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃            alkenyl, and H;        -   R₈ is selected from the group consisting of C₃₋₆ carbocycle            and heterocycle;        -   R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆            alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆            carbocycle and heterocycle;        -   each R is independently selected from the group consisting            of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;    -   each R′ is independently selected from the group consisting of        C₁₋₁₈ s alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₅ alkyl and C₃₋₁₅ alkenyl;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each Y is independently a C₃₋₆ carbocycle;    -   each X is independently selected from the group consisting of F,        Cl, Br, and I; and    -   m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and        wherein when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or        —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5,        or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is        1 or 2.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IA):

-   -   or its N-oxide, or a salt or isomer thereof, wherein 1 is        selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8,        and 9; M₁ is a bond or M′; R₄ is hydrogen, unsubstituted C₁₋₃        alkyl, or —(CH₂)_(n)Q, in which Q is    -   OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂,        —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ are        independently selected    -   from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—,        —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and        R₂ and R₃ are independently selected from the group consisting        of H, C1-14 alkyl, and C₂₋₁₄ alkenyl. For example, m is 5, 7,        or 9. For example, Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂. For        example, Q is —N(R)C(O)R, or —N(R)S(O)₂R.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (IB):

or its N-oxide, or a salt or isomer thereof in which all variables areas defined herein. For example, m is selected from 5, 6, 7, 8, and 9;

-   -   R₄ is hydrogen, unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in        which Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R,        —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,        —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and        M′ are independently selected    -   from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—,        —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and        R₂ and R₃ are independently selected from the group consisting        of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl. For example, m is 5, 7,        or 9. For example, Q is OH, —NHC(S)N(R)₂, or —NHC(O)N(R)₂. For        example, Q is —N(R)C(O)R, or —N(R)S(O)₂R.

In certain embodiments, a subset of compounds of Formula (I) includesthose of Formula (II):

or its N-oxide, or a salt or isomer thereof, wherein 1 is selected from1, 2, 3, 4, and 5; M₁ is a bond or M′; R₄ is hydrogen, unsubstitutedC₁₋₃ alkyl, or —(CH₂)_(n)Q, in which n is 2, 3, or 4, and Q is

-   -   OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,        —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂,        —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ are        independently selected    -   from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—,        —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and        R₂ and R₃ are independently selected from the group consisting        of H, C1-14 alkyl, and C₂₋₁₄ alkenyl.

In one embodiment, the compounds of Formula (I) are of Formula (IIa),

or their N-oxides, or salts or isomers thereof, wherein R₄ is asdescribed herein.

In another embodiment, the compounds of Formula (I) are of Formula(IIb),

or their N-oxides, or salts or isomers thereof, wherein R₄ is asdescribed herein.

In another embodiment, the compounds of Formula (I) are of Formula (IIc)or (IIe):

or their N-oxides, or salts or isomers thereof, wherein R₄ is asdescribed herein.

In another embodiment, the compounds of Formula (I) are of Formula(IIf):

or their N-oxides, or salts or isomers thereof,

wherein M is —C(O)O— or —OC(O)—, M″ is C₁₋₆ alkyl or C₂₋₆ alkenyl, R₂and R₃ are independently selected from the group consisting of C₅₋₁₄alkyl and C₅₋₁₄ alkenyl, and n is selected from 2, 3, and 4.

In a further embodiment, the compounds of Formula (I) are of Formula(IId),

or their N-oxides, or salts or isomers thereof, wherein n is 2, 3, or 4;and m, R′, R″, and R₂ through R₆ are as described herein. For example,each of R₂ and R₃ may be independently selected from the groupconsisting of C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl.

In a further embodiment, the compounds of Formula (I) are of Formula(IIg),

or their N-oxides, or salts or isomers thereof, wherein 1 is selectedfrom 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; M₁ is abond or M′; M and M′ are independently selected

-   -   from —C(O)O—, —OC(O)—, —OC(O)-M″-C(O)O—, —C(O)N(R′)—,        —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and        R₂ and R₃ are independently selected from the group consisting        of H, C1-14 alkyl, and C₂₋₁₄ alkenyl. For example, M″ is C₁₋₆        alkyl (e.g., C1-4 alkyl) or C₂₋₆ alkenyl (e.g. C₂₋₄ alkenyl).        For example, R₂ and R₃ are independently selected from the group        consisting of C₅₋₁₄ alkyl and C₅₋₁₄ alkenyl.

In some embodiments, the ionizable lipids are one or more of thecompounds described in U.S. Application Nos. 62/220,091, 62/252,316,62/253,433, 62/266,460, 62/333,557, 62/382,740, 62/393,940, 62/471,937,62/471,949, 62/475,140, and 62/475,166, and PCT Application No.PCT/US2016/052352.

In some embodiments, the ionizable lipids are selected from Compounds1-280 described in U.S. Application No. 62/475,166.

In some embodiments, the ionizable lipid is

or a salt thereof.

In some embodiments, the ionizable lipid is

or a salt thereof.

In some embodiments, the ionizable lipid is

or a salt thereof.

In some embodiments, the ionizable lipid is

or a salt thereof.

The central amine moiety of a lipid according to Formula (I), (IA),(IB), (II), (IIa), (IIb), (IIc), (IId), (IIe), (IIf), or (IIg) may beprotonated at a physiological pH. Thus, a lipid may have a positive orpartial positive charge at physiological pH. Such lipids may be referredto as cationic or ionizable (amino)lipids. Lipids may also bezwitterionic, i.e., neutral molecules having both a positive and anegative charge.

In some aspects, the ionizable lipids of the present disclosure may beone or more of compounds of formula (III),

or salts or isomers thereof, wherein

W is

ring A is

-   -   t is 1 or 2;    -   A₁ and A₂ are each independently selected from CH or N;    -   Z is CH₂ or absent wherein when Z is CH₂, the dashed lines (1)        and (2) each represent a single bond; and when Z is absent, the        dashed lines (1) and (2) are both absent;    -   R₁, R₂, R₃, R₄, and R₅ are independently selected from the group        consisting of C₅₋₂₀ alkyl, C₅₋₂₀ alkenyl, —R″MR′, —R*YR″, —YR″,        and —R*OR″;    -   R_(X1) and R_(X2) are each independently H or C₁₋₃ alkyl;    -   each M is independently selected from the group consisting    -   of —C(O)O—, —OC(O)—, —OC(O)O—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—,        —C(S)—, —C(S)S—, —SC(S)—,    -   —C_(H)(OH)—, —P(O)(OR′)O—, —S(O)₂—, —C(O)S—, —SC(O)—, an aryl        group, and a heteroaryl group;    -   M* is C1-C6 alkyl,    -   W¹ and W² are each independently selected from the group        consisting of —O— and —N(R₆)—;    -   each R₆ is independently selected from the group consisting of H        and C₁₅ alkyl;    -   X¹, X², and X³ are independently selected from the group        consisting of a bond, —CH₂—, —(CH₂)₂—, —CHR—, —CHY—, —C(O)—,        —C(O)O—, —OC(O)—, —(CH₂)_(n)—C(O)—, —C(O)—(CH₂)_(n)—,        —(CH₂)_(n)—C(O)O—, —OC(O)—(CH₂)_(n)—, —(CH₂)_(n)—OC(O)—,        —C(O)O—(CH₂)_(n)—, —C_(H)(OH)—, —C(S)—, and —C_(H)(SH)—;    -   each Y is independently a C₃₋₆ carbocycle;    -   each R* is independently selected from the group consisting of        C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl;    -   each R is independently selected from the group consisting of        C₁₋₃ alkyl and a C₃₋₆ carbocycle;    -   each R′ is independently selected from the group consisting of        C1-12 alkyl, C₂₋₁₂ alkenyl, and H;    -   each R″ is independently selected from the group consisting of        C₃₋₁₂ alkyl, C₃₋₁₂ alkenyl and —R*MR′; and    -   n is an integer from 1-6;

wherein when ring A is

then

-   -   i) at least one of X¹, X², and X³ is not —CH₂—; and/or    -   ii) at least one of R₁, R₂, R₃, R₄, and R₅ is —R″MR′.

In some embodiments, the compound is of any of formulae (IIIa1)-(IIIa8):

In some embodiments, the ionizable lipids are one or more of thecompounds described in U.S. Application Nos. 62/271,146, 62/338,474,62/413,345, and 62/519,826, and PCT Application No. PCT/US2016/068300.

In some embodiments, the ionizable lipids are selected from Compounds1-156 described in U.S. Application No. 62/519,826.

In some embodiments, the ionizable lipids are selected from Compounds1-16, 42-66, 68-76, and 78-156 described in U.S. Application No.62/519,826.

In some embodiments, the ionizable lipid is

or a salt thereof.

The central amine moiety of a lipid according to Formula (III), (IIIa1),(IIIa2), (IIIa3), (IIIa4), (IIIa5), (IIIa6), (IIIa7), or (IIIa8) may beprotonated at a physiological pH. Thus, a lipid may have a positive orpartial positive charge at physiological pH. Such lipids may be referredto as cationic or ionizable (amino)lipids. Lipids may also bezwitterionic, i.e., neutral molecules having both a positive and anegative charge.

Phospholipids

The lipid composition of the lipid nanoparticle composition disclosedherein can comprise one or more phospholipids, for example, one or moresaturated or (poly)unsaturated phospholipids or a combination thereof.In general, phospholipids comprise a phospholipid moiety and one or morefatty acid moieties.

A phospholipid moiety can be selected, for example, from thenon-limiting group consisting of phosphatidyl choline, phosphatidylethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidicacid, 2-lysophosphatidyl choline, and a sphingomyelin.

A fatty acid moiety can be selected, for example, from the non-limitinggroup consisting of lauric acid, myristic acid, myristoleic acid,palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleicacid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid,arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoicacid, and docosahexaenoic acid.

Particular phospholipids can facilitate fusion to a membrane. Forexample, a cationic phospholipid can interact with one or morenegatively charged phospholipids of a membrane (e.g., a cellular orintracellular membrane). Fusion of a phospholipid to a membrane canallow one or more elements (e.g., a therapeutic agent) of alipid-containing composition (e.g., LNPs) to pass through the membranepermitting, e.g., delivery of the one or more elements to a targettissue.

Non-natural phospholipid species including natural species withmodifications and substitutions including branching, oxidation,cyclization, and alkynes are also contemplated. For example, aphospholipid can be functionalized with or cross-linked to one or morealkynes (e.g., an alkenyl group in which one or more double bonds isreplaced with a triple bond). Under appropriate reaction conditions, analkyne group can undergo a copper-catalyzed cycloaddition upon exposureto an azide. Such reactions can be useful in functionalizing a lipidbilayer of a nanoparticle composition to facilitate membrane permeationor cellular recognition or in conjugating a nanoparticle composition toa useful component such as a targeting or imaging moiety (e.g., a dye).

Phospholipids include, but are not limited to, glycerophospholipids suchas phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.Phospholipids also include phosphosphingolipid, such as sphingomyelin.

In some embodiments, a phospholipid of the invention comprises1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC),1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC),1-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine(OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine,1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE),1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG),sphingomyelin, and mixtures thereof.

In certain embodiments, a phospholipid useful or potentially useful inthe present invention is an analog or variant of DSPC. In certainembodiments, a phospholipid useful or potentially useful in the presentinvention is a compound of Formula (IV):

-   -   or a salt thereof, wherein:    -   each R¹ is independently optionally substituted alkyl; or        optionally two R¹ are joined together with the intervening atoms        to form optionally substituted monocyclic carbocyclyl or        optionally substituted monocyclic heterocyclyl; or optionally        three R¹ are joined together with the intervening atoms to form        optionally substituted bicyclic carbocyclyl or optionally        substitute bicyclic heterocyclyl;    -   n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   A is of the formula:

-   -   each instance of L² is independently a bond or optionally        substituted C₁₋₆ alkylene, wherein one methylene unit of the        optionally substituted C₁₋₆ alkylene is optionally replaced with        0, N(R^(N)), S, C(O), C(O)N(R^(N)), NR^(N)C(O), C(O)O, OC(O),        OC(O)O, OC(O)N(R^(N)), NR^(N)C(O), or NR^(N)C(O)N(R^(N));    -   each instance of R² is independently optionally substituted        C₁₋₃₀ alkyl, optionally substituted C₁₋₃₀ alkenyl, or optionally        substituted C₁₋₃₀ alkynyl; optionally wherein one or more        methylene units of R² are independently replaced with optionally        substituted carbocyclylene, optionally substituted        heterocyclylene, optionally substituted arylene, optionally        substituted heteroarylene, N(R^(N)), O, S, C(O), C(O)N(R^(N)),        NR^(N)C(O), NR^(N)C(O)N(R^(N)), C(O)O, OC(O), OC(O)O,        OC(O)N(R^(N)), NR^(N)C(O)O, C(O)S, SC(O), C(═NR^(N)),        C(═NR^(N))N(R^(N)), —NR^(N)C(═NR^(N)), NR^(N)C(═NR^(N))N(R^(N)),        C(S), C(S)N(R^(N)), NR^(N)C(S), NR^(N)C(S)N(R^(N)), S(O)—OS(O),        S(O)O, OS(O)O, OS(O)₂, S(O)₂O, OS(O)₂O, N(R^(N))S(O),        S(O)N(R^(N)), —N(R^(N))S(O)N(R^(N)), OS(O)N(R^(N)),        N(R^(N))S(O)O, S(O)₂, N(R^(N))S(O)₂, S(O)₂N(R^(N)),        —N(R^(N))S(O)₂N(R^(N)), OS(O)₂N(R^(N)), or N(R^(N))S(O)₂O;    -   each instance of R^(N) is independently hydrogen, optionally        substituted alkyl, or a nitrogen protecting group;

Ring B is optionally substituted carbocyclyl, optionally substitutedheterocyclyl, optionally substituted aryl, or optionally substitutedheteroaryl; and

-   -   p is 1 or 2;    -   provided that the compound is not of the formula:

wherein each instance of R₂ is independently unsubstituted alkyl,unsubstituted alkenyl, or unsubstituted alkynyl.

In some embodiments, the phospholipids may be one or more of thephospholipids described in U.S. Application No. 62/520,530.

i) Phospholipid Head Modifications

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified phospholipid head (e.g., amodified choline group). In certain embodiments, a phospholipid with amodified head is DSPC, or analog thereof, with a modified quaternaryamine. For example, in embodiments of Formula (IV), at least one of R¹is not methyl. In certain embodiments, at least one of R¹ is nothydrogen or methyl. In certain embodiments, the compound of Formula (IV)is of one of the following formulae:

-   -   or a salt thereof, wherein:    -   each t is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;    -   each u is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and    -   each v is independently 1, 2, or 3.

In certain embodiments, a compound of Formula (IV) is of Formula (IV-a):

or a salt thereof.

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a cyclic moiety in place of theglyceride moiety. In certain embodiments, a phospholipid useful in thepresent invention is DSPC, or analog thereof, with a cyclic moiety inplace of the glyceride moiety. In certain embodiments, the compound ofFormula (IV) is of Formula (IV-b):

or a salt thereof.

(ii) Phospholipid Tail Modifications

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified tail. In certain embodiments,a phospholipid useful or potentially useful in the present invention isDSPC, or analog thereof, with a modified tail. As described herein, a“modified tail” may be a tail with shorter or longer aliphatic chains,aliphatic chains with branching introduced, aliphatic chains withsubstituents introduced, aliphatic chains wherein one or more methylenesare replaced by cyclic or heteroatom groups, or any combination thereof.For example, in certain embodiments, the compound of (IV) is of Formula(IV-a), or a salt thereof, wherein at least one instance of R² is eachinstance of R² is optionally substituted C₁₋₃₀ alkyl, wherein one ormore methylene units of R₂ are independently replaced with optionallysubstituted carbocyclylene, optionally substituted heterocyclylene,optionally substituted arylene, optionally substituted heteroarylene,N(R^(N)), O, S, C(O), C(O)N(R^(N)), NR^(N)C(O), NR^(N)C(O)N(R^(N)),C(O)O, OC(O), OC(O)O, OC(O)N(R^(N)NR^(N)C(O)O, C(O)S, SC(O), C(═NR^(N)),C(═NR^(N))N(R^(N)), NR^(N)C(═NR^(N)), NR^(N)C(═NR^(N))N(R^(N)), C(S),C(S)N(R^(N)), NR^(N)C(S), NR^(N)C(S)N(R^(N)), S(O), OS(O), S(O)O,OS(O)O, OS(O)₂, —S(O)₂O, OS(O)₂O, N(R^(N))S(O), S(O)N(R^(N)),N(R^(N))S(O)N(R^(N)), OS(O)N(R^(N)), N(R^(N))S(O)O, S(O)₂,N(R^(N))S(O)₂, S(O)₂N(R^(N)), N(R^(N))S(O)₂N(R^(N)), OS(O)₂N(R^(N)), orN(R^(N))S(O)₂O.

In certain embodiments, the compound of Formula (IV) is of Formula(IV-c):

-   -   or a salt thereof, wherein:    -   each x is independently an integer between 0-30, inclusive; and    -   each instance is G is independently selected from the group        consisting of optionally substituted carbocyclylene, optionally        substituted heterocyclylene, optionally substituted arylene,        optionally substituted heteroarylene, N(R^(N)), O, S, C(O),        C(O)N(R^(N)), NR^(N)C(O), —NR^(N)C(O)N(R^(N)), C(O)O, OC(O),        OC(O)O, OC(O)N(R^(N)), NR^(N)C(O), C(O)S, SC(O), —C(═NR^(N)),        C(═NR^(N))N(R^(N)), NR^(N)C(═NR^(N)), NR^(N)C(═NR^(N))N(R^(N)),        C(S), C(S)N(R^(N)), NR^(N)C(S), NR^(N)C(S)N(R^(N)), S(O), OS(O),        S(O)O, OS(O)O, OS(O)₂, S(O)₂O, OS(O)₂O, N(R^(N))S(O),        —S(O)N(R^(N)), N(R^(N))S(O)N(R^(N)), OS(O)N(R^(N)),        N(R^(N))S(O)O, S(O)₂, N(R^(N))S(O)₂, S(O)₂N(R^(N)),        N(R^(N))S(O)₂N(R^(N)), OS(O)₂N(R^(N)), or N(R^(N))S(O)₂O. Each        possibility represents a separate embodiment of the present        invention.

In certain embodiments, a phospholipid useful or potentially useful inthe present invention comprises a modified phosphocholine moiety,wherein the alkyl chain linking the quaternary amine to the phosphorylgroup is not ethylene (e.g., n is not 2). Therefore, in certainembodiments, a phospholipid useful or potentially useful in the presentinvention is a compound of Formula (IV), wherein n is 1, 3, 4, 5, 6, 7,8, 9, or 10. For example, in certain embodiments, a compound of Formula(IV) is of one of the following formulae:

or a salt thereof.

Alternative Lipids

In certain embodiments, an alternative lipid is used in place of aphospholipid of the present disclosure.

In certain embodiments, an alternative lipid of the invention is oleicacid.

In certain embodiments, the alternative lipid is one of the following:

Structural Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more structural lipids. As used herein, the term“structural lipid” refers to sterols and also to lipids containingsterol moieties.

Incorporation of structural lipids in the lipid nanoparticle may helpmitigate aggregation of other lipids in the particle. Structural lipidscan be selected from the group including but not limited to,cholesterol, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid,alpha-tocopherol, hopanoids, phytosterols, steroids, and mixturesthereof. In some embodiments, the structural lipid is a sterol. Asdefined herein, “sterols” are a subgroup of steroids consisting ofsteroid alcohols. In certain embodiments, the structural lipid is asteroid. In certain embodiments, the structural lipid is cholesterol. Incertain embodiments, the structural lipid is an analog of cholesterol.In certain embodiments, the structural lipid is alpha-tocopherol.

In some embodiments, the structural lipids may be one or more of thestructural lipids described in U.S. Application No. 62/520,530.

Polyethylene Glycol (PEG)-Lipids

The lipid composition of a pharmaceutical composition disclosed hereincan comprise one or more a polyethylene glycol (PEG) lipid.

As used herein, the term “PEG-lipid” refers to polyethylene glycol(PEG)-modified lipids. Non-limiting examples of PEG-lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modifieddialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipidsare also referred to as PEGylated lipids. For example, a PEG lipid canbe PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPElipid.

In some embodiments, the PEG-lipid includes, but not limited to1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl,PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG),PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA).

In one embodiment, the PEG-lipid is selected from the group consistingof a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidicacid, a PEG-modified ceramide, a PEG-modified dialkylamine, aPEG-modified diacylglycerol, a PEG-modified dialkylglycerol, andmixtures thereof.

In some embodiments, the lipid moiety of the PEG-lipids includes thosehaving lengths of from about C₁₄ to about C₂₂, preferably from about C₁₄to about C₁₆. In some embodiments, a PEG moiety, for example a mPEG-NH₂,has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons.In one embodiment, the PEG-lipid is PEG2k-DMG.

In one embodiment, the lipid nanoparticles described herein can comprisea PEG lipid which is a non-diffusible PEG. Non-limiting examples ofnon-diffusible PEGs include PEG-DSG and PEG-DSPE.

PEG-lipids are known in the art, such as those described in U.S. Pat.No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which areincorporated herein by reference in their entirety.

In general, some of the other lipid components (e.g., PEG lipids) ofvarious formulae, described herein may be synthesized as describedInternational Patent Application No. PCT/US2016/000129, filed Dec. 10,2016, entitled “Compositions and Methods for Delivery of TherapeuticAgents,” which is incorporated by reference in its entirety.

The lipid component of a lipid nanoparticle composition may include oneor more molecules comprising polyethylene glycol, such as PEG orPEG-modified lipids. Such species may be alternately referred to asPEGylated lipids. A PEG lipid is a lipid modified with polyethyleneglycol. A PEG lipid may be selected from the non-limiting groupincluding PEG-modified phosphatidylethanolamines, PEG-modifiedphosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols, andmixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments the PEG-modified lipids are a modified form of PEGDMG. PEG-DMG has the following structure:

In one embodiment, PEG lipids useful in the present invention can bePEGylated lipids described in International Publication No.WO2012099755, the contents of which is herein incorporated by referencein its entirety. Any of these exemplary PEG lipids described herein maybe modified to comprise a hydroxyl group on the PEG chain. In certainembodiments, the PEG lipid is a PEG-OH lipid. As generally definedherein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylatedlipid”) is a PEGylated lipid having one or more hydroxyl (—OH) groups onthe lipid. In certain embodiments, the PEG-OH lipid includes one or morehydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH orhydroxy-PEGylated lipid comprises an —OH group at the terminus of thePEG chain. Each possibility represents a separate embodiment of thepresent invention.

In certain embodiments, a PEG lipid useful in the present invention is acompound of Formula (V). Provided herein are compounds of Formula (V):

-   -   or salts thereof, wherein:    -   R₃ is —OR^(O);    -   R^(O) is hydrogen, optionally substituted alkyl, or an oxygen        protecting group;    -   r is an integer between 1 and 100, inclusive;    -   L¹ is optionally substituted C₁₋₁₀ alkylene, wherein at least        one methylene of the optionally substituted C₁₋₁₀ alkylene is        independently replaced with optionally substituted        carbocyclylene, optionally substituted heterocyclylene,        optionally substituted arylene, optionally substituted        heteroarylene, O, N(R^(N)), S, C(O), C(O)N(R^(N)), NR^(N)C(O),        C(O)O, OC(O), OC(O)O, OC(O)N(R^(N)), NR^(N)C(O)O, or        NR^(N)C(O)N(R^(N));    -   D is a moiety obtained by click chemistry or a moiety cleavable        under physiological conditions;    -   m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

A is of the formula:

-   -   each instance of L² is independently a bond or optionally        substituted C₁₋₆ alkylene, wherein one methylene unit of the        optionally substituted C₁₋₆ alkylene is optionally replaced with        O, N(R^(N)), S, C(O), C(O)N(R^(N)), NR^(N)C(O), C(O)O, OC(O),        OC(O)O, OC(O)N(R^(N)), NR^(N)C(O)O, or NR^(N)C(O)N(R^(N));    -   each instance of R² is independently optionally substituted        C₁₋₃₀ alkyl, optionally substituted C₁₋₃₀ alkenyl, or optionally        substituted C₁₋₃₀ alkynyl; optionally wherein one or more        methylene units of R² are independently replaced with optionally        substituted carbocyclylene, optionally substituted        heterocyclylene, optionally substituted arylene, optionally        substituted heteroarylene, N(R^(N)), O, S, C(O), C(O)N(R^(N)),        NR^(N)C(O), NR^(N)C(O)N(R^(N)), C(O)O, OC(O), OC(O)O,        OC(O)N(R^(N)), NR^(N)C(O)O, C(O)S, SC(O), C(═NR^(N)),        C(═NR^(N))N(R^(N)), —NR^(N)C(═NR^(N)), NR^(N)C(═NR^(N))N(R^(N)),        C(S), C(S)N(R^(N)), NR^(N)C(S), NR^(N)C(S)N(R^(N)), S(O),        —OS(O), S(O)O, OS(O)O, OS(O)₂, S(O)₂O, OS(O)₂O, N(R^(N))S(O),        S(O)N(R^(N)), —N(R^(N))S(O)N(R^(N)), OS(O)N(R^(N)),        N(R^(N))S(O)O, S(O)₂, N(R^(N))S(O)₂, S(O)₂N(R^(N)),        —N(R^(N))S(O)₂N(R^(N)), OS(O)₂N(R^(N)), or N(R^(N))S(O)₂O;    -   each instance of R^(N) is independently hydrogen, optionally        substituted alkyl, or a nitrogen protecting group;    -   Ring B is optionally substituted carbocyclyl, optionally        substituted heterocyclyl, optionally substituted aryl, or        optionally substituted heteroaryl; and    -   p is 1 or 2.

In certain embodiments, the compound of Formula (V) is a PEG-OH lipid(i.e., R³ is —OR^(O), and R^(O) is hydrogen). In certain embodiments,the compound of Formula (V) is of Formula (V-OH):

or a salt thereof.

In certain embodiments, a PEG lipid useful in the present invention is aPEGylated fatty acid. In certain embodiments, a PEG lipid useful in thepresent invention is a compound of Formula (VI). Provided herein arecompounds of Formula (VI)

-   -   or a salts thereof, wherein:    -   R³ is —OR^(O);    -   R^(O) is hydrogen, optionally substituted alkyl or an oxygen        protecting group;    -   r is an integer between 1 and 100, inclusive;    -   R⁵ is optionally substituted C₁₀₋₄₀ alkyl, optionally        substituted C₁₀₋₄₀ alkenyl, or optionally substituted C₁₀₋₄₀        alkynyl; and optionally one or more methylene groups of R⁵ are        replaced with optionally substituted carbocyclylene, optionally        substituted heterocyclylene, optionally substituted arylene,        optionally substituted heteroarylene, N(R^(N)), O, S, C(O),        C(O)N(R^(N)), —NR^(N)C(O), NR^(N)C(O)N(R^(N)), C(O)O, OC(O),        OC(O)O, OC(O)N(R^(N)), NR^(N)C(O)O C(O)S, —SC(O), C(═NR^(N)),        C(═NR^(N))N(R^(N)), NR^(N)C(═NR^(N)), NR^(N)C(═NR^(N))N(R^(N)),        C(S), C(S)N(R^(N)), —NR^(N)C(S), NR^(N)C(S)N(R^(N)), S(O),        OS(O), S(O)O, OS(O)O, OS(O)₂, S(O)₂O, OS(O)₂O, —N(R^(N))S(O),        S(O)N(R^(N)), N(R^(N))S(O)N(R^(N)), OS(O)N(R^(N)),        N(R^(N))S(O)O, S(O)₂, N(R^(N))S(O)₂, S(O)₂N(R^(N)),        N(R^(N))S(O)₂N(R^(N)), OS(O)₂N(R^(N)), or N(R^(N))S(O)₂O; and    -   each instance of R^(N) is independently hydrogen, optionally        substituted alkyl, or a nitrogen protecting group.

In certain embodiments, the compound of Formula (VI) is of Formula(VI-OH):

or a salt thereof. In some embodiments, r is 45.

In yet other embodiments the compound of Formula (VI) is:

or a salt thereof.

In one embodiment, the compound of Formula (VI) is

In some aspects, the lipid composition of the pharmaceuticalcompositions disclosed herein does not comprise a PEG-lipid.

In some embodiments, the PEG-lipids may be one or more of the PEG lipidsdescribed in U.S. Application No. 62/520,530.

In some embodiments, a PEG lipid of the invention comprises aPEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid,a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modifieddiacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. Insome embodiments, the PEG-modified lipid is PEG-DMG, PEG-c-DOMG (alsoreferred to as PEG-DOMG), PEG-DSG and/or PEG-DPG.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of any of Formula I, II or III, a phospholipid comprisingDSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of any of Formula I, II or III, a phospholipid comprisingDSPC, a structural lipid, and a PEG lipid comprising a compound havingFormula VI.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of Formula I, II or III, a phospholipid comprising acompound having Formula IV, a structural lipid, and the PEG lipidcomprising a compound having Formula V or VI.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of Formula I, II or III, a phospholipid comprising acompound having Formula IV, a structural lipid, and the PEG lipidcomprising a compound having Formula V or VI.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of Formula I, II or III, a phospholipid having FormulaIV, a structural lipid, and a PEG lipid comprising a compound havingFormula VI.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of

and a PEG lipid comprising Formula VI.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of

and an alternative lipid comprising oleic acid.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of

an alternative lipid comprising oleic acid, a structural lipidcomprising cholesterol, and a PEG lipid comprising a compound havingFormula VI.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of

-   -   a phospholipid comprising DOPE, a structural lipid comprising        cholesterol, and a PEG lipid comprising a compound having        Formula VI.

In some embodiments, a LNP of the invention comprises an ionizablecationic lipid of

a phospholipid comprising DOPE, a structural lipid comprisingcholesterol, and a PEG lipid comprising a compound having Formula VII.

In some embodiments, a LNP of the invention comprises an N:P ratio offrom about 2:1 to about 30:1.

In some embodiments, a LNP of the invention comprises an N:P ratio ofabout 6:1.

In some embodiments, a LNP of the invention comprises an N:P ratio ofabout 3:1.

In some embodiments, a LNP of the invention comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of from about 10:1 toabout 100:1.

In some embodiments, a LNP of the invention comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of about 20:1.

In some embodiments, a LNP of the invention comprises a wt/wt ratio ofthe ionizable cationic lipid component to the RNA of about 10:1.

In some embodiments, a LNP of the invention has a mean diameter fromabout 50 nm to about 150 nm.

In some embodiments, a LNP of the invention has a mean diameter fromabout 70 nm to about 120 nm.

As used herein, the term “alkyl”, “alkyl group”, or “alkylene” means alinear or branched, saturated hydrocarbon including one or more carbonatoms (e.g., one, two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms), which is optionallysubstituted. The notation “C1-14 alkyl” means an optionally substitutedlinear or branched, saturated hydrocarbon including 1-14 carbon atoms.Unless otherwise specified, an alkyl group described herein refers toboth unsubstituted and substituted alkyl groups.

As used herein, the term “alkenyl”, “alkenyl group”, or “alkenylene”means a linear or branched hydrocarbon including two or more carbonatoms (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms) and at least onedouble bond, which is optionally substituted. The notation “C₂₋₁₄alkenyl” means an optionally substituted linear or branched hydrocarbonincluding 2-14 carbon atoms and at least one carbon-carbon double bond.An alkenyl group may include one, two, three, four, or morecarbon-carbon double bonds. For example, C18 alkenyl may include one ormore double bonds. A C18 alkenyl group including two double bonds may bea linoleyl group. Unless otherwise specified, an alkenyl group describedherein refers to both unsubstituted and substituted alkenyl groups.

As used herein, the term “alkynyl”, “alkynyl group”, or “alkynylene”means a linear or branched hydrocarbon including two or more carbonatoms (e.g., two, three, four, five, six, seven, eight, nine, ten,eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen,eighteen, nineteen, twenty, or more carbon atoms) and at least onecarbon-carbon triple bond, which is optionally substituted. The notation“C₂₋₁₄ alkynyl” means an optionally substituted linear or branchedhydrocarbon including 2-14 carbon atoms and at least one carbon-carbontriple bond. An alkynyl group may include one, two, three, four, or morecarbon-carbon triple bonds. For example, C18 alkynyl may include one ormore carbon-carbon triple bonds. Unless otherwise specified, an alkynylgroup described herein refers to both unsubstituted and substitutedalkynyl groups.

As used herein, the term “carbocycle” or “carbocyclic group” means anoptionally substituted mono- or multi-cyclic system including one ormore rings of carbon atoms. Rings may be three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, or twenty membered rings. The notation“C3-6 carbocycle” means a carbocycle including a single ring having 3-6carbon atoms. Carbocycles may include one or more carbon-carbon doubleor triple bonds and may be non-aromatic or aromatic (e.g., cycloalkyl oraryl groups). Examples of carbocycles include cyclopropyl, cyclopentyl,cyclohexyl, phenyl, naphthyl, and 1,2-dihydronaphthyl groups. The term“cycloalkyl” as used herein means a non-aromatic carbocycle and may ormay not include any double or triple bond. Unless otherwise specified,carbocycles described herein refers to both unsubstituted andsubstituted carbocycle groups, i.e., optionally substituted carbocycles.

As used herein, the term “heterocycle” or “heterocyclic group” means anoptionally substituted mono- or multi-cyclic system including one ormore rings, where at least one ring includes at least one heteroatom.Heteroatoms may be, for example, nitrogen, oxygen, or sulfur atoms.Rings may be three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, or fourteen membered rings. Heterocycles may includeone or more double or triple bonds and may be non-aromatic or aromatic(e.g., heterocycloalkyl or heteroaryl groups). Examples of heterocyclesinclude imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl,thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl,isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl,furyl, tetrahydrofuryl, thiophenyl, pyridinyl, piperidinyl, quinolyl,and isoquinolyl groups. The term “heterocycloalkyl” as used herein meansa non-aromatic heterocycle and may or may not include any double ortriple bond. Unless otherwise specified, heterocycles described hereinrefers to both unsubstituted and substituted heterocycle groups, i.e.,optionally substituted heterocycles.

As used herein, the term “heteroalkyl”, “heteroalkenyl”, or“heteroalkynyl”, refers respectively to an alkyl, alkenyl, alkynylgroup, as defined herein, which further comprises one or more (e.g., 1,2, 3, or 4) heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon,phosphorus) wherein the one or more heteroatoms is inserted betweenadjacent carbon atoms within the parent carbon chain and/or one or moreheteroatoms is inserted between a carbon atom and the parent molecule,i.e., between the point of attachment. Unless otherwise specified,heteroalkyls, heteroalkenyls, or heteroalkynyls described herein refersto both unsubstituted and substituted heteroalkyls, heteroalkenyls, orheteroalkynyls, i.e., optionally substituted heteroalkyls,heteroalkenyls, or heteroalkynyls.

As used herein, a “biodegradable group” is a group that may facilitatefaster metabolism of a lipid in a mammalian entity. A biodegradablegroup may be selected from the group consisting of, but is not limited

-   -   to, —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—,

—C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, an arylgroup, and a heteroaryl group. As used herein, an “aryl group” is anoptionally substituted carbocyclic group including one or more aromaticrings. Examples of aryl groups include phenyl and naphthyl groups. Asused herein, a “heteroaryl group” is an optionally substitutedheterocyclic group including one or more aromatic rings. Examples ofheteroaryl groups include pyrrolyl, furyl, thiophenyl, imidazolyl,oxazolyl, and thiazolyl. Both aryl and heteroaryl groups may beoptionally substituted. For example, M and M′ can be selected from thenon-limiting group consisting of optionally substituted phenyl, oxazole,and thiazole. In the formulas herein, M and M′ can be independentlyselected from the list of biodegradable groups above. Unless otherwisespecified, aryl or heteroaryl groups described herein refers to bothunsubstituted and substituted groups, i.e., optionally substituted arylor heteroaryl groups.

Alkyl, alkenyl, and cyclyl (e.g., carbocyclyl and heterocyclyl) groupsmay be optionally substituted unless otherwise specified. Optionalsubstituents may be selected from the group consisting of, but are notlimited to, a halogen atom (e.g., a chloride, bromide, fluoride, oriodide group), a carboxylic acid (e.g., —C(O)OH), an alcohol (e.g., ahydroxyl, —OH), an ester (e.g., —C(O)OR—OC(O)R), an aldehyde (e.g.,—C(O)H), a carbonyl (e.g., —C(O)R, alternatively represented by C═O), anacyl halide (e.g., —C(O)X, in which X is a halide selected from bromide,fluoride, chloride, and iodide), a carbonate (e.g., —OC(O)OR), an alkoxy(e.g., —OR), an acetal (e.g., —C(OR)₂R″″, in which each OR are alkoxygroups that can be the same or different and R″″ is an alkyl or alkenylgroup), a phosphate (e.g., P(O)43-), a thiol (e.g., —SH), a sulfoxide(e.g., —S(O)R), a sulfinic acid (e.g., —S(O)OH), a sulfonic acid (e.g.,—S(O)2OH), a thial (e.g., —C(S)H), a sulfate (e.g., S(O)42-), a sulfonyl(e.g., —S(O)2-), an amide (e.g., —C(O)NR2, or —N(R)C(O)R), an azido(e.g., —N3), a nitro (e.g., —NO2), a cyano (e.g., —CN), an isocyano(e.g., —NC), an acyloxy (e.g., —OC(O)R), an amino (e.g., —NR2, —NRH, or—NH2), a carbamoyl (e.g., —OC(O)NR2, —OC(O)NRH, or —OC(O)NH2), asulfonamide (e.g., —S(O)2NR2, —S(O)2NRH, —S(O)2NH2, —N(R)S(O)2R,—N(H)S(O)2R, —N(R)S(O)2H, or —N(H)S(O)2H), an alkyl group, an alkenylgroup, and a cyclyl (e.g., carbocyclyl or heterocyclyl) group. In any ofthe preceding, R is an alkyl or alkenyl group, as defined herein. Insome embodiments, the substituent groups themselves may be furthersubstituted with, for example, one, two, three, four, five, or sixsubstituents as defined herein. For example, a C₁₋₆ alkyl group may befurther substituted with one, two, three, four, five, or sixsubstituents as described herein.

Compounds of the disclosure that contain nitrogens can be converted toN-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the disclosure. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N□O or N+—O—). Furthermore, inother instances, the nitrogens in the compounds of the disclosure can beconverted to N-hydroxy or N-alkoxy compounds. For example, N-hydroxycompounds can be prepared by oxidation of the parent amine by anoxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

About, approximately: As used herein, the terms “approximately” and“about,” as applied to one or more values of interest, refer to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value). For example, when used in the contextof an amount of a given compound in a lipid component of a nanoparticlecomposition, “about” may mean +/−10% of the recited value. For instance,a nanoparticle composition including a lipid component having about 40%of a given compound may include 30-50% of the compound.

As used herein, the term “compound,” is meant to include all isomers andisotopes of the structure depicted. “Isotopes” refers to atoms havingthe same atomic number but different mass numbers resulting from adifferent number of neutrons in the nuclei. For example, isotopes ofhydrogen include tritium and deuterium. Further, a compound, salt, orcomplex of the present disclosure can be prepared in combination withsolvent or water molecules to form solvates and hydrates by routinemethods.

(vi) Other Lipid Composition Components

The lipid composition of a pharmaceutical composition disclosed hereincan include one or more components in addition to those described above.For example, the lipid composition can include one or more permeabilityenhancer molecules, carbohydrates, polymers, surface altering agents(e.g., surfactants), or other components. For example, a permeabilityenhancer molecule can be a molecule described by U.S. Patent ApplicationPublication No. 2005/0222064. Carbohydrates can include simple sugars(e.g., glucose) and polysaccharides (e.g., glycogen and derivatives andanalogs thereof).

A polymer can be included in and/or used to encapsulate or partiallyencapsulate a pharmaceutical composition disclosed herein (e.g., apharmaceutical composition in lipid nanoparticle form). A polymer can bebiodegradable and/or biocompatible. A polymer can be selected from, butis not limited to, polyamines, polyethers, polyamides, polyesters,polycarbamates, polyureas, polycarbonates, polystyrenes, polyimides,polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates.

The ratio between the lipid composition and the polynucleotide range canbe from about 10:1 to about 60:1 (wt/wt).

In some embodiments, the ratio between the lipid composition and thepolynucleotide can be about 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1, 21:1, 22:1, 23:1, 24:1, 25:1, 26:1, 27:1, 28:1,29:1, 30:1, 31:1, 32:1, 33:1, 34:1, 35:1, 36:1, 37:1, 38:1, 39:1, 40:1,41:1, 42:1, 43:1, 44:1, 45:1, 46:1, 47:1, 48:1, 49:1, 50:1, 51:1, 52:1,53:1, 54:1, 55:1, 56:1, 57:1, 58:1, 59:1 or 60:1 (wt/wt). In someembodiments, the wt/wt ratio of the lipid composition to thepolynucleotide encoding a therapeutic agent is about 20:1 or about 15:1.

In some embodiments, the pharmaceutical composition disclosed herein cancontain more than one polypeptides. For example, a pharmaceuticalcomposition disclosed herein can contain two or more polynucleotides(e.g., RNA, e.g., mRNA).

In one embodiment, the lipid nanoparticles described herein can comprisepolynucleotides (e.g., mRNA) in a lipid:polynucleotide weight ratio of5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 or70:1, or a range or any of these ratios such as, but not limited to, 5:1to about 10:1, from about 5:1 to about 15:1, from about 5:1 to about20:1, from about 5:1 to about 25:1, from about 5:1 to about 30:1, fromabout 5:1 to about 35:1, from about 5:1 to about 40:1, from about 5:1 toabout 45:1, from about 5:1 to about 50:1, from about 5:1 to about 55:1,from about 5:1 to about 60:1, from about 5:1 to about 70:1, from about10:1 to about 15:1, from about 10:1 to about 20:1, from about 10:1 toabout 25:1, from about 10:1 to about 30:1, from about 10:1 to about35:1, from about 10:1 to about 40:1, from about 10:1 to about 45:1, fromabout 10:1 to about 50:1, from about 10:1 to about 55:1, from about 10:1to about 60:1, from about 10:1 to about 70:1, from about 15:1 to about20:1, from about 15:1 to about 25:1, from about 15:1 to about 30:1, fromabout 15:1 to about 35:1, from about 15:1 to about 40:1, from about 15:1to about 45:1, from about 15:1 to about 50:1, from about 15:1 to about55:1, from about 15:1 to about 60:1 or from about 15:1 to about 70:1.

In one embodiment, the lipid nanoparticles described herein can comprisethe polynucleotide in a concentration from approximately 0.1 mg/ml to 2mg/ml such as, but not limited to, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml, 1.0 mg/ml,1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6 mg/ml, 1.7mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml or greater than 2.0 mg/ml.

(vii) Nanoparticle Compositions

In some embodiments, the pharmaceutical compositions disclosed hereinare formulated as lipid nanoparticles (LNP). Accordingly, the presentdisclosure also provides nanoparticle compositions comprising (i) alipid composition comprising a delivery agent such as compound asdescribed herein, and (ii) a polynucleotide encoding an anti-CHIKVantibody polypeptide. In such nanoparticle composition, the lipidcomposition disclosed herein can encapsulate the polynucleotide encodingan anti-CHIKV antibody polypeptide.

Nanoparticle compositions are typically sized on the order ofmicrometers or smaller and can include a lipid bilayer. Nanoparticlecompositions encompass lipid nanoparticles (LNPs), liposomes (e.g.,lipid vesicles), and lipoplexes. For example, a nanoparticle compositioncan be a liposome having a lipid bilayer with a diameter of 500 nm orless.

Nanoparticle compositions include, for example, lipid nanoparticles(LNPs), liposomes, and lipoplexes. In some embodiments, nanoparticlecompositions are vesicles including one or more lipid bilayers. Incertain embodiments, a nanoparticle composition includes two or moreconcentric bilayers separated by aqueous compartments. Lipid bilayerscan be functionalized and/or crosslinked to one another. Lipid bilayerscan include one or more ligands, proteins, or channels.

In one embodiment, a lipid nanoparticle comprises an ionizable lipid, astructural lipid, a phospholipid, and mRNA. In some embodiments, the LNPcomprises an ionizable lipid, a PEG-modified lipid, a sterol and astructural lipid. In some embodiments, the LNP has a molar ratio ofabout 20-60% ionizable lipid: about 5-25% structural lipid: about 25-55%sterol; and about 0.5-15% PEG-modified lipid.

In some embodiments, the LNP has a polydispersity value of less than0.4. In some embodiments, the LNP has a net neutral charge at a neutralpH. In some embodiments, the LNP has a mean diameter of 50-150 nm. Insome embodiments, the LNP has a mean diameter of 80-100 nm.

As generally defined herein, the term “lipid” refers to a small moleculethat has hydrophobic or amphiphilic properties. Lipids may be naturallyoccurring or synthetic. Examples of classes of lipids include, but arenot limited to, fats, waxes, sterol-containing metabolites, vitamins,fatty acids, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids, and polyketides, and prenol lipids. In some instances,the amphiphilic properties of some lipids leads them to form liposomes,vesicles, or membranes in aqueous media.

In some embodiments, a lipid nanoparticle (LNP) may comprise anionizable lipid. As used herein, the term “ionizable lipid” has itsordinary meaning in the art and may refer to a lipid comprising one ormore charged moieties. In some embodiments, an ionizable lipid may bepositively charged or negatively charged. An ionizable lipid may bepositively charged, in which case it can be referred to as “cationiclipid”. In certain embodiments, an ionizable lipid molecule may comprisean amine group, and can be referred to as an ionizable amino lipid. Asused herein, a “charged moiety” is a chemical moiety that carries aformal electronic charge, e.g., monovalent (+1, or −1), divalent (+2, or−2), trivalent (+3, or −3), etc. The charged moiety may be anionic(i.e., negatively charged) or cationic (i.e., positively charged).Examples of positively-charged moieties include amine groups (e.g.,primary, secondary, and/or tertiary amines), ammonium groups, pyridiniumgroup, guanidine groups, and imidizolium groups. In a particularembodiment, the charged moieties comprise amine groups. Examples ofnegatively-charged groups or precursors thereof, include carboxylategroups, sulfonate groups, sulfate groups, phosphonate groups, phosphategroups, hydroxyl groups, and the like. The charge of the charged moietymay vary, in some cases, with the environmental conditions, for example,changes in pH may alter the charge of the moiety, and/or cause themoiety to become charged or uncharged. In general, the charge density ofthe molecule may be selected as desired.

It should be understood that the terms “charged” or “charged moiety”does not refer to a “partial negative charge” or “partial positivecharge” on a molecule. The terms “partial negative charge” and “partialpositive charge” are given its ordinary meaning in the art. A “partialnegative charge” may result when a functional group comprises a bondthat becomes polarized such that electron density is pulled toward oneatom of the bond, creating a partial negative charge on the atom. Thoseof ordinary skill in the art will, in general, recognize bonds that canbecome polarized in this way.

In some embodiments, the ionizable lipid is an ionizable amino lipid,sometimes referred to in the art as an “ionizable cationic lipid”. Inone embodiment, the ionizable amino lipid may have a positively chargedhydrophilic head and a hydrophobic tail that are connected via a linkerstructure.

In addition to these, an ionizable lipid may also be a lipid including acyclic amine group.

In one embodiment, the ionizable lipid may be selected from, but notlimited to, a ionizable lipid described in International PublicationNos. WO2013086354 and WO2013116126; the contents of each of which areherein incorporated by reference in their entirety.

In yet another embodiment, the ionizable lipid may be selected from, butnot limited to, formula CLI-CLXXXXII of U.S. Pat. No. 7,404,969; each ofwhich is herein incorporated by reference in their entirety.

In one embodiment, the lipid may be a cleavable lipid such as thosedescribed in International Publication No. WO2012170889, hereinincorporated by reference in its entirety. In one embodiment, the lipidmay be synthesized by methods known in the art and/or as described inInternational Publication Nos. WO2013086354; the contents of each ofwhich are herein incorporated by reference in their entirety.

Nanoparticle compositions can be characterized by a variety of methods.For example, microscopy (e.g., transmission electron microscopy orscanning electron microscopy) can be used to examine the morphology andsize distribution of a nanoparticle composition. Dynamic lightscattering or potentiometry (e.g., potentiometric titrations) can beused to measure zeta potentials. Dynamic light scattering can also beutilized to determine particle sizes. Instruments such as the ZetasizerNano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) can alsobe used to measure multiple characteristics of a nanoparticlecomposition, such as particle size, polydispersity index, and zetapotential. The size of the nanoparticles can help counter biologicalreactions such as, but not limited to, inflammation, or can increase thebiological effect of the polynucleotide. As used herein, “size” or “meansize” in the context of nanoparticle compositions refers to the meandiameter of a nanoparticle composition.

In one embodiment, the polynucleotide encoding an anti-CHIKV antibodypolypeptide are formulated in lipid nanoparticles having a diameter fromabout 10 to about 100 nm such as, but not limited to, about 10 to about20 nm, about 10 to about 30 nm, about to about 40 nm, about 10 to about50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 toabout 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm,about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 toabout 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about50 to about 60 nm, about 50 to about 70 nm, about 50 to about 80 nm,about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 toabout 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/orabout 90 to about 100 nm.

In one embodiment, the nanoparticles have a diameter from about 10 to500 nm. In one embodiment, the nanoparticle has a diameter greater than100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm,greater than 300 nm, greater than 350 nm, greater than 400 nm, greaterthan 450 nm, greater than 500 nm, greater than 550 nm, greater than 600nm, greater than 650 nm, greater than 700 nm, greater than 750 nm,greater than 800 nm, greater than 850 nm, greater than 900 nm, greaterthan 950 nm or greater than 1000 nm.

In some embodiments, the largest dimension of a nanoparticle compositionis 1 μm or shorter (e.g., 1 μm, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm,400 nm, 300 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, orshorter).

A nanoparticle composition can be relatively homogenous. Apolydispersity index can be used to indicate the homogeneity of ananoparticle composition, e.g., the particle size distribution of thenanoparticle composition. A small (e.g., less than 0.3) polydispersityindex generally indicates a narrow particle size distribution. Ananoparticle composition can have a polydispersity index from about 0 toabout 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08,0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20,0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersityindex of a nanoparticle composition disclosed herein can be from about0.10 to about 0.20.

The zeta potential of a nanoparticle composition can be used to indicatethe electrokinetic potential of the composition. For example, the zetapotential can describe the surface charge of a nanoparticle composition.Nanoparticle compositions with relatively low charges, positive ornegative, are generally desirable, as more highly charged species caninteract undesirably with cells, tissues, and other elements in thebody. In some embodiments, the zeta potential of a nanoparticlecomposition disclosed herein can be from about −10 mV to about +20 mV,from about −10 mV to about +15 mV, from about 10 mV to about +10 mV,from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, fromabout −10 mV to about −5 mV, from about −5 mV to about +20 mV, fromabout −5 mV to about +15 mV, from about −5 mV to about +10 mV, fromabout −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0mV to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV toabout +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about+20 mV, from about +5 mV to about +15 mV, or from about +5 mV to about+10 mV.

In some embodiments, the zeta potential of the lipid nanoparticles canbe from about 0 mV to about 100 mV, from about 0 mV to about 90 mV, fromabout 0 mV to about 80 mV, from about 0 mV to about 70 mV, from about 0mV to about 60 mV, from about 0 mV to about 50 mV, from about 0 mV toabout 40 mV, from about 0 mV to about 30 mV, from about 0 mV to about 20mV, from about 0 mV to about 10 mV, from about 10 mV to about 100 mV,from about 10 mV to about 90 mV, from about 10 mV to about 80 mV, fromabout mV to about 70 mV, from about 10 mV to about 60 mV, from about 10mV to about 50 mV, from about 10 mV to about 40 mV, from about 10 mV toabout 30 mV, from about 10 mV to about 20 mV, from about 20 mV to about100 mV, from about 20 mV to about 90 mV, from about 20 mV to about 80mV, from about 20 mV to about 70 mV, from about 20 mV to about 60 mV,from about 20 mV to about 50 mV, from about 20 mV to about 40 mV, fromabout 20 mV to about 30 mV, from about 30 mV to about 100 mV, from about30 mV to about 90 mV, from about 30 mV to about 80 mV, from about 30 mVto about 70 mV, from about 30 mV to about 60 mV, from about 30 mV toabout 50 mV, from about 30 mV to about mV, from about 40 mV to about 100mV, from about 40 mV to about 90 mV, from about mV to about 80 mV, fromabout 40 mV to about 70 mV, from about 40 mV to about 60 mV, and fromabout 40 mV to about 50 mV. In some embodiments, the zeta potential ofthe lipid nanoparticles can be from about 10 mV to about 50 mV, fromabout 15 mV to about 45 mV, from about 20 mV to about 40 mV, and fromabout 25 mV to about 35 mV. In some embodiments, the zeta potential ofthe lipid nanoparticles can be about 10 mV, about 20 mV, about 30 mV,about 40 mV, about 50 mV, about 60 mV, about 70 mV, about 80 mV, about90 mV, and about 100 mV.

The term “encapsulation efficiency” of a polynucleotide describes theamount of the polynucleotide that is encapsulated by or otherwiseassociated with a nanoparticle composition after preparation, relativeto the initial amount provided. As used herein, “encapsulation” canrefer to complete, substantial, or partial enclosure, confinement,surrounding, or encasement.

Encapsulation efficiency is desirably high (e.g., close to 100%). Theencapsulation efficiency can be measured, for example, by comparing theamount of the polynucleotide in a solution containing the nanoparticlecomposition before and after breaking up the nanoparticle compositionwith one or more organic solvents or detergents.

Fluorescence can be used to measure the amount of free polynucleotide ina solution. For the nanoparticle compositions described herein, theencapsulation efficiency of a polynucleotide can be at least 50%, forexample 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulationefficiency can be at least 80%. In certain embodiments, theencapsulation efficiency can be at least 90%.

The amount of a polynucleotide present in a pharmaceutical compositiondisclosed herein can depend on multiple factors such as the size of thepolynucleotide, desired target and/or application, or other propertiesof the nanoparticle composition as well as on the properties of thepolynucleotide.

For example, the amount of an mRNA useful in a nanoparticle compositioncan depend on the size (expressed as length, or molecular mass),sequence, and other characteristics of the mRNA. The relative amounts ofa polynucleotide in a nanoparticle composition can also vary.

The relative amounts of the lipid composition and the polynucleotidepresent in a lipid nanoparticle composition of the present disclosurecan be optimized according to considerations of efficacy andtolerability. For compositions including an mRNA as a polynucleotide,the N:P ratio can serve as a useful metric.

As the N:P ratio of a nanoparticle composition controls both expressionand tolerability, nanoparticle compositions with low N:P ratios andstrong expression are desirable. N:P ratios vary according to the ratioof lipids to RNA in a nanoparticle composition.

In general, a lower N:P ratio is preferred. The one or more RNA, lipids,and amounts thereof can be selected to provide an N:P ratio from about2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1,12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. Incertain embodiments, the N:P ratio can be from about 2:1 to about 8:1.In other embodiments, the N:P ratio is from about 5:1 to about 8:1. Incertain embodiments, the N:P ratio is between 5:1 and 6:1. In onespecific aspect, the N:P ratio is about is about 5.67:1.

In addition to providing nanoparticle compositions, the presentdisclosure also provides methods of producing lipid nanoparticlescomprising encapsulating a polynucleotide. Such method comprises usingany of the pharmaceutical compositions disclosed herein and producinglipid nanoparticles in accordance with methods of production of lipidnanoparticles known in the art. See, e.g., Wang et al. (2015) “Deliveryof oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev.87:68-80; Silva et al. (2015) “Delivery Systems for Biopharmaceuticals.Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol. 16:940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles andNanostructured Lipid Carriers: Structure, Preparation and Application”Adv. Pharm. Bull. 5:305-13; Silva et al. (2015) “Lipid nanoparticles forthe delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302,and references cited therein.

21. Other Delivery Agents

a. Liposomes, Lipoplexes, and Lipid Nanoparticles

In some embodiments, the compositions or formulations of the presentdisclosure comprise a delivery agent, e.g., a liposome, a lioplexes, alipid nanoparticle, or any combination thereof. The polynucleotidesdescribed herein (e.g., a polynucleotide comprising a nucleotidesequence encoding an anti-CHIKV antibody polypeptide) can be formulatedusing one or more liposomes, lipoplexes, or lipid nanoparticles.Liposomes, lipoplexes, or lipid nanoparticles can be used to improve theefficacy of the polynucleotides directed protein production as theseformulations can increase cell transfection by the polynucleotide;and/or increase the translation of encoded protein. The liposomes,lipoplexes, or lipid nanoparticles can also be used to increase thestability of the polynucleotides.

Liposomes are artificially-prepared vesicles that can primarily becomposed of a lipid bilayer and can be used as a delivery vehicle forthe administration of pharmaceutical formulations. Liposomes can be ofdifferent sizes. A multilamellar vesicle (MLV) can be hundreds ofnanometers in diameter, and can contain a series of concentric bilayersseparated by narrow aqueous compartments. A small unicellular vesicle(SUV) can be smaller than 50 nm in diameter, and a large unilamellarvesicle (LUV) can be between 50 and 500 nm in diameter. Liposome designcan include, but is not limited to, opsonins or ligands to improve theattachment of liposomes to unhealthy tissue or to activate events suchas, but not limited to, endocytosis. Liposomes can contain a low or ahigh pH value in order to improve the delivery of the pharmaceuticalformulations.

The formation of liposomes can depend on the pharmaceutical formulationentrapped and the liposomal ingredients, the nature of the medium inwhich the lipid vesicles are dispersed, the effective concentration ofthe entrapped substance and its potential toxicity, any additionalprocesses involved during the application and/or delivery of thevesicles, the optimal size, polydispersity and the shelf-life of thevesicles for the intended application, and the batch-to-batchreproducibility and scale up production of safe and efficient liposomalproducts, etc.

As a non-limiting example, liposomes such as synthetic membrane vesiclescan be prepared by the methods, apparatus and devices described in U.S.Pub. Nos. US20130177638, US20130177637, US20130177636, US20130177635,US20130177634, US20130177633, US20130183375, US20130183373, andUS20130183372. In some embodiments, the polynucleotides described hereincan be encapsulated by the liposome and/or it can be contained in anaqueous core that can then be encapsulated by the liposome as describedin, e.g., Intl. Pub. Nos. W2012031046, W2012031043, W2012030901,WO2012006378, and WO2013086526; and U.S. Pub. Nos. US20130189351,US20130195969 and US20130202684. Each of the references in hereinincorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a cationic oil-in-water emulsion where the emulsionparticle comprises an oil core and a cationic lipid that can interactwith the polynucleotide anchoring the molecule to the emulsion particle.In some embodiments, the polynucleotides described herein can beformulated in a water-in-oil emulsion comprising a continuoushydrophobic phase in which the hydrophilic phase is dispersed. Exemplaryemulsions can be made by the methods described in Intl. Pub. Nos.WO2012006380 and WO201087791, each of which is herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a lipid-polycation complex. The formation of thelipid-polycation complex can be accomplished by methods as described in,e.g., U.S. Pub. No. US20120178702. As a non-limiting example, thepolycation can include a cationic peptide or a polypeptide such as, butnot limited to, polylysine, polyornithine and/or polyarginine and thecationic peptides described in Intl. Pub. No. WO2012013326 or U.S. Pub.No. US20130142818. Each of the references is herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a lipid nanoparticle (LNP) such as those described inIntl. Pub. Nos. W2013123523, WO2012170930, WO2011127255 andWO2008103276; and U.S. Pub. No. US20130171646, each of which is hereinincorporated by reference in its entirety.

Lipid nanoparticle formulations typically comprise one or more lipids.In some embodiments, the lipid is an ionizable lipid (e.g., an ionizableamino lipid), sometimes referred to in the art as an “ionizable cationiclipid”. In some embodiments, lipid nanoparticle formulations furthercomprise other components, including a phospholipid, a structural lipid,and a molecule capable of reducing particle aggregation, for example aPEG or PEG-modified lipid.

Exemplary ionizable lipids include, but not limited to, any one ofCompounds 1-342 disclosed herein, DLin-MC3-DMA (MC3), DLin-DMA, DLenDMA,DLin-D-DMA, DLin-K-DMA, DLin-M-C2-DMA, DLin-K-DMA, DLin-KC2-DMA,DLin-KC3-DMA, DLin-KC4-DMA, DLin-C2K-DMA, DLin-MP-DMA, DODMA, 98N12-5,C12-200, DLin-C-DAP, DLin-DAC, DLinDAP, DLinAP, DLin-EG-DMA,DLin-2-DMAP, KL10, KL22, KL25, Octyl-CLinDMA, Octyl-CLinDMA (2R),Octyl-CLinDMA (2S), and any combination thereof. Other exemplaryionizable lipids include,(13Z,16Z)—N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine (L608),(20Z,23Z)—N,N-dimethylnonacosa-20,23-dien-10-amine,(17Z,20Z)—N,N-dimemylhexacosa-17,20-dien-9-amine,(16Z,19Z)—N5N-dimethylpentacosa-16,19-dien-8-amine,(13Z,16Z)—N,N-dimethyldocosa-13,16-dien-5-amine,(12Z,15Z)—N,N-dimethylhenicosa-12,15-dien-4-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-6-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-7-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-10-amine,(15Z,18Z)—N,N-dimethyltetracosa-15,18-dien-5-amine,(14Z,17Z)—N,N-dimethyltricosa-14,17-dien-4-amine,(19Z,22Z)—N,N-dimeihyloctacosa-19,22-dien-9-amine,(18Z,21Z)—N,N-dimethylheptacosa-18,21-dien-8-amine,(17Z,20Z)—N,N-dimethylhexacosa-17,20-dien-7-amine,(16Z,19Z)—N,N-dimethylpentacosa-16,19-dien-6-amine,(22Z,25Z)—N,N-dimethylhentriaconta-22,25-dien-10-amine,(21Z,24Z)—N,N-dimethyltriaconta-21,24-dien-9-amine,(18Z)—N,N-dimetylheptacos-18-en-10-amine,(17Z)—N,N-dimethylhexacos-17-en-9-amine,(19Z,22Z)—N,N-dimethyloctacosa-19,22-dien-7-amine,N,N-dimethylheptacosan-10-amine,(20Z,23Z)—N-ethyl-N-methylnonacosa-20,23-dien-10-amine,1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine,(20Z)—N,N-dimethylheptacos-20-en-10-amine, (15Z)—N,N-dimethyleptacos-15-en-10-amine, (14Z)—N,N-dimethylnonacos-14-en-10-amine,(17Z)—N,N-dimethylnonacos-17-en-10-amine,(24Z)—N,N-dimethyltritriacont-24-en-10-amine,(20Z)—N,N-dimethylnonacos-20-en-10-amine,(22Z)—N,N-dimethylhentriacont-22-en-10-amine,(16Z)—N,N-dimethylpentacos-16-en-8-amine,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,N,N-dimethyl-1-[(S,2R)-2-octylcyclopropyl] eptadecan-8-amine,1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine,N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine,N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine,1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine,1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine,N,N-dimethyl-1-[(S,2R)-2-octylcyclopropyl]pentadecan-8-amine,R—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,S—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine,(2S)—N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine,1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine,(2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine;(2S)—N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine,(2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine,(2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine,1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,(2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine,1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine,(2R)—N,N-dimethyl-H(1-metoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,(2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine,N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine,N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine,and (11E,20Z,23Z)—N,N-dimethylnonacosa-11,20,2-trien-10-amine, and anycombination thereof.

Phospholipids include, but are not limited to, glycerophospholipids suchas phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines,phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids.Phospholipids also include phosphosphingolipid, such as sphingomyelin.In some embodiments, the phospholipids are DLPC, DMPC, DOPC, DPPC, DSPC,DUPC, 18:0 Diether PC, DLnPC, DAPC, DHAPC, DOPE, 4ME 16:0 PE, DSPE,DLPE, DLnPE, DAPE, DHAPE, DOPG, and any combination thereof. In someembodiments, the phospholipids are MPPC, MSPC, PMPC, PSPC, SMPC, SPPC,DHAPE, DOPG, and any combination thereof. In some embodiments, theamount of phospholipids (e.g., DSPC) in the lipid composition rangesfrom about 1 mol % to about 20 mol %.

The structural lipids include sterols and lipids containing sterolmoieties. In some embodiments, the structural lipids includecholesterol, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid,alpha-tocopherol, and mixtures thereof. In some embodiments, thestructural lipid is cholesterol. In some embodiments, the amount of thestructural lipids (e.g., cholesterol) in the lipid composition rangesfrom about 20 mol % to about 60 mol %.

The PEG-modified lipids include PEG-modified phosphatidylethanolamineand phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 orPEG-CerC20), PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Such lipids are also referred to asPEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG DMPE, PEG-DPPC, or a PEG-DSPE lipid. In some embodiments,the PEG-lipid are 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol(PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE),PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl,PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoylphosphatidylethanolamine (PEG-DPPE), orPEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments,the PEG moiety has a size of about 1000, 2000, 5000, 10,000, 15,000 or20,000 daltons. In some embodiments, the amount of PEG-lipid in thelipid composition ranges from about 0 mol % to about 5 mol %.

In some embodiments, the LNP formulations described herein canadditionally comprise a permeability enhancer molecule. Non-limitingpermeability enhancer molecules are described in U.S. Pub. No.US20050222064, herein incorporated by reference in its entirety.

The LNP formulations can further contain a phosphate conjugate. Thephosphate conjugate can increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatescan be made by the methods described in, e.g., Intl. Pub. No.WO2013033438 or U.S. Pub. No. US20130196948. The LNP formulation canalso contain a polymer conjugate (e.g., a water soluble conjugate) asdescribed in, e.g., U.S. Pub. Nos. US20130059360, US20130196948, andUS20130072709. Each of the references is herein incorporated byreference in its entirety.

The LNP formulations can comprise a conjugate to enhance the delivery ofnanoparticles of the present invention in a subject. Further, theconjugate can inhibit phagocytic clearance of the nanoparticles in asubject. In some embodiments, the conjugate can be a “self” peptidedesigned from the human membrane protein CD47 (e.g., the “self”particles described by Rodriguez et al, Science 2013 339, 971-975,herein incorporated by reference in its entirety). As shown by Rodriguezet al. the self peptides delayed macrophage-mediated clearance ofnanoparticles which enhanced delivery of the nanoparticles.

The LNP formulations can comprise a carbohydrate carrier. As anon-limiting example, the carbohydrate carrier can include, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin (e.g., Intl. Pub. No.WO2012109121, herein incorporated by reference in its entirety).

The LNP formulations can be coated with a surfactant or polymer toimprove the delivery of the particle. In some embodiments, the LNP canbe coated with a hydrophilic coating such as, but not limited to, PEGcoatings and/or coatings that have a neutral surface charge as describedin U.S. Pub. No. US20130183244, herein incorporated by reference in itsentirety.

The LNP formulations can be engineered to alter the surface propertiesof particles so that the lipid nanoparticles can penetrate the mucosalbarrier as described in U.S. Pat. No. 8,241,670 or Intl. Pub. No.WO2013110028, each of which is herein incorporated by reference in itsentirety.

The LNP engineered to penetrate mucus can comprise a polymeric material(i.e., a polymeric core) and/or a polymer-vitamin conjugate and/or atri-block co-polymer. The polymeric material can include, but is notlimited to, polyamines, polyethers, polyamides, polyesters,polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides,polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates.

LNP engineered to penetrate mucus can also include surface alteringagents such as, but not limited to, polynucleotides, anionic proteins(e.g., bovine serum albumin), surfactants (e.g., cationic surfactantssuch as for example dimethyldioctadecyl-ammonium bromide), sugars orsugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g.,heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g.,N-acetylcysteine, mugwort, bromelain, papain, clerodendrum,acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol,sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin04 domase alfa, neltenexine, erdosteine) and various DNases includingrhDNase.

In some embodiments, the mucus penetrating LNP can be a hypotonicformulation comprising a mucosal penetration enhancing coating. Theformulation can be hypotonic for the epithelium to which it is beingdelivered. Non-limiting examples of hypotonic formulations can be foundin, e.g., Intl. Pub. No. W2013110028, herein incorporated by referencein its entirety.

In some embodiments, the polynucleotide described herein is formulatedas a lipoplex, such as, without limitation, the ATUPLEX™ system, theDACC system, the DBTC system and other siRNA-lipoplex technology fromSilence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT®(Cambridge, MA), and polyethylenimine (PEI) or protamine-based targetedand non-targeted delivery of nucleic acids (Aleku et al. Cancer Res.2008 68:9788-9798; Strumberg et al. Int J Clin Pharmacol Ther 201250:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al.,Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 201023:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293 Weide et al. JImmunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188;Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011J. Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005,23:709-717; Peer et al., Proc Natl Acad Sci USA. 2007 6; 104:4095-4100;deFougerolles Hum Gene Ther. 2008 19:125-132; all of which areincorporated herein by reference in its entirety).

In some embodiments, the polynucleotides described herein are formulatedas a solid lipid nanoparticle (SLN), which can be spherical with anaverage diameter between 10 to 1000 nm. SLN possess a solid lipid corematrix that can solubilize lipophilic molecules and can be stabilizedwith surfactants and/or emulsifiers. Exemplary SLN can be those asdescribed in Intl. Pub. No. WO2013105101, herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated for controlled release and/or targeted delivery. As usedherein, “controlled release” refers to a pharmaceutical composition orcompound release profile that conforms to a particular pattern ofrelease to effect a therapeutic outcome. In one embodiment, thepolynucleotides can be encapsulated into a delivery agent describedherein and/or known in the art for controlled release and/or targeteddelivery. As used herein, the term “encapsulate” means to enclose,surround or encase. As it relates to the formulation of the compounds ofthe invention, encapsulation can be substantial, complete or partial.The term “substantially encapsulated” means that at least greater than50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, or greater than 99% of thepharmaceutical composition or compound of the invention can be enclosed,surrounded or encased within the delivery agent. “Partiallyencapsulation” means that less than 10, 10, 20, 30, 40 50 or less of thepharmaceutical composition or compound of the invention can be enclosed,surrounded or encased within the delivery agent.

Advantageously, encapsulation can be determined by measuring the escapeor the activity of the pharmaceutical composition or compound of theinvention using fluorescence and/or electron micrograph. For example, atleast 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99,99.9, or greater than 99% of the pharmaceutical composition or compoundof the invention are encapsulated in the delivery agent.

In some embodiments, the polynucleotides described herein can beencapsulated in a therapeutic nanoparticle, referred to herein as“therapeutic nanoparticle polynucleotides.” Therapeutic nanoparticlescan be formulated by methods described in, e.g., Intl. Pub. Nos.WO2010005740, WO2010030763, WO2010005721, WO2010005723, andWO2012054923; and U.S. Pub. Nos. US20110262491, US20100104645,US20100087337, US20100068285, US20110274759, US20100068286,US20120288541, US20120140790, US20130123351 and US20130230567; and U.S.Pat. Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211, each of whichis herein incorporated by reference in its entirety.

In some embodiments, the therapeutic nanoparticle polynucleotide can beformulated for sustained release. As used herein, “sustained release”refers to a pharmaceutical composition or compound that conforms to arelease rate over a specific period of time. The period of time caninclude, but is not limited to, hours, days, weeks, months and years. Asa non-limiting example, the sustained release nanoparticle of thepolynucleotides described herein can be formulated as disclosed in Intl.Pub. No. W2010075072 and U.S. Pub. Nos. US20100216804, US20110217377,US20120201859 and US20130150295, each of which is herein incorporated byreference in their entirety.

In some embodiments, the therapeutic nanoparticle polynucleotide can beformulated to be target specific, such as those described in Intl. Pub.Nos. W2008121949, WO2010005726, WO2010005725, WO2011084521 andWO2011084518; and U.S. Pub. Nos. US20100069426, US20120004293 andUS20100104655, each of which is herein incorporated by reference in itsentirety.

The LNPs can be prepared using microfluidic mixers or micromixers.Exemplary microfluidic mixers can include, but are not limited to, aslit interdigital micromixer including, but not limited to thosemanufactured by Microinnova (Allerheiligen bei Wildon, Austria) and/or astaggered herringbone micromixer (SHM) (see Zhigaltsev et al.,“Bottom-up design and synthesis of limit size lipid nanoparticle systemswith aqueous and triglyceride cores using millisecond microfluidicmixing,” Langmuir 28:3633-40 (2012); Belliveau et al., “Microfluidicsynthesis of highly potent limit-size lipid nanoparticles for in vivodelivery of siRNA,” Molecular Therapy-Nucleic Acids. 1:e37 (2012); Chenet al., “Rapid discovery of potent siRNA-containing lipid nanoparticlesenabled by controlled microfluidic formulation,” J. Am. Chem. Soc.134(16):6948-51 (2012); each of which is herein incorporated byreference in its entirety). Exemplary micromixers include SlitInterdigital Microstructured Mixer (SIMM-V2) or a Standard SlitInterdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging-jet(IJMM,) from the Institut für Mikrotechnik Mainz GmbH, Mainz Germany. Insome embodiments, methods of making LNP using SHM further comprisemixing at least two input streams wherein mixing occurs bymicrostructure-induced chaotic advection (MICA). According to thismethod, fluid streams flow through channels present in a herringbonepattern causing rotational flow and folding the fluids around eachother. This method can also comprise a surface for fluid mixing whereinthe surface changes orientations during fluid cycling. Methods ofgenerating LNPs using SHM include those disclosed in U.S. Pub. Nos.US20040262223 and US20120276209, each of which is incorporated herein byreference in their entirety.

In some embodiments, the polynucleotides described herein can beformulated in lipid nanoparticles using microfluidic technology (seeWhitesides, George M., “The Origins and the Future of Microfluidics,”Nature 442: 368-373 (2006); and Abraham et al., “Chaotic Mixer forMicrochannels,” Science 295: 647-651 (2002); each of which is hereinincorporated by reference in its entirety). In some embodiments, thepolynucleotides can be formulated in lipid nanoparticles using amicromixer chip such as, but not limited to, those from HarvardApparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK). Amicromixer chip can be used for rapid mixing of two or more fluidstreams with a split and recombine mechanism.

In some embodiments, the polynucleotides described herein can beformulated in lipid nanoparticles having a diameter from about 1 nm toabout 100 nm such as, but not limited to, about 1 nm to about 20 nm,from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, fromabout 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm toabout 90 nm, from about 5 nm to about from 100 nm, from about 5 nm toabout 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm,from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, fromabout 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 20nm, about 10 to about nm, about 10 to about 40 nm, about 10 to about 50nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 toabout 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm,about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 toabout 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm,about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about100 nm, about 80 to about 90 nm, about 80 to about 100 nm and/or about90 to about 100 nm.

In some embodiments, the lipid nanoparticles can have a diameter fromabout 10 to 500 nm. In one embodiment, the lipid nanoparticle can have adiameter greater than 100 nm, greater than 150 nm, greater than 200 nm,greater than 250 nm, greater than 300 nm, greater than 350 nm, greaterthan 400 nm, greater than 450 nm, greater than 500 nm, greater than 550nm, greater than 600 nm, greater than 650 nm, greater than 700 nm,greater than 750 nm, greater than 800 nm, greater than 850 nm, greaterthan 900 nm, greater than 950 nm or greater than 1000 nm.

In some embodiments, the polynucleotides can be delivered using smallerLNPs. Such particles can comprise a diameter from below 0.1 μm up to 100nm such as, but not limited to, less than 0.1 μm, less than 1.0 μm, lessthan 5 μm, less than 10 μm, less than 15 um, less than 20 um, less than25 um, less than 30 um, less than 35 um, less than 40 um, less than 50um, less than 55 um, less than 60 um, less than 65 um, less than 70 um,less than 75 um, less than 80 um, less than 85 um, less than 90 um, lessthan 95 um, less than 100 um, less than 125 um, less than 150 um, lessthan 175 um, less than 200 um, less than 225 um, less than 250 um, lessthan 275 um, less than 300 um, less than 325 um, less than 350 um, lessthan 375 um, less than 400 um, less than 425 um, less than 450 um, lessthan 475 um, less than 500 um, less than 525 um, less than 550 um, lessthan 575 um, less than 600 um, less than 625 um, less than 650 um, lessthan 675 um, less than 700 um, less than 725 um, less than 750 um, lessthan 775 um, less than 800 um, less than 825 um, less than 850 um, lessthan 875 um, less than 900 um, less than 925 um, less than 950 um, orless than 975 um.

The nanoparticles and microparticles described herein can begeometrically engineered to modulate macrophage and/or the immuneresponse. The geometrically engineered particles can have varied shapes,sizes and/or surface charges to incorporate the polynucleotidesdescribed herein for targeted delivery such as, but not limited to,pulmonary delivery (see, e.g., Intl. Pub. No. WO2013082111, hereinincorporated by reference in its entirety). Other physical features thegeometrically engineering particles can include, but are not limited to,fenestrations, angled arms, asymmetry and surface roughness, charge thatcan alter the interactions with cells and tissues.

In some embodiment, the nanoparticles described herein are stealthnanoparticles or target-specific stealth nanoparticles such as, but notlimited to, those described in U.S. Pub. No. US20130172406, hereinincorporated by reference in its entirety. The stealth ortarget-specific stealth nanoparticles can comprise a polymeric matrix,which can comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), poly cyanoacrylates,polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates, or combinationsthereof.

b. Lipidoids

In some embodiments, the compositions or formulations of the presentdisclosure comprise a delivery agent, e.g., a lipidoid. Thepolynucleotides described herein (e.g., a polynucleotide comprising anucleotide sequence encoding an anti-CHIKV antibody polypeptide) can beformulated with lipidoids. Complexes, micelles, liposomes or particlescan be prepared containing these lipidoids and therefore to achieve aneffective delivery of the polynucleotide, as judged by the production ofan encoded protein, following the injection of a lipidoid formulationvia localized and/or systemic routes of administration. Lipidoidcomplexes of polynucleotides can be administered by various meansincluding, but not limited to, intravenous, intramuscular, orsubcutaneous routes.

The synthesis of lipidoids is described in literature (see Mahon et al.,Bioconjug. Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al.,Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc NatlAcad Sci USA. 2011 108:12996-3001; all of which are incorporated hereinin their entireties).

Formulations with the different lipidoids, including, but not limited topenta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride(TETA-5LAP; also known as 98N12-5, see Murugaiah et al., AnalyticalBiochemistry, 401:61 (2010)), C12-200 (including derivatives andvariants), and MD1, can be tested for in vivo activity. The lipidoid“98N12-5” is disclosed by Akinc et al., Mol Ther. 2009 17:872-879. Thelipidoid “C12-200” is disclosed by Love et al., Proc Natl Acad Sci USA.2010 107:1864-1869 and Liu and Huang, Molecular Therapy. 2010 669-670.Each of the references is herein incorporated by reference in itsentirety.

In one embodiment, the polynucleotides described herein can beformulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids can beprepared by the methods described in U.S. Pat. No. 8,450,298 (hereinincorporated by reference in its entirety).

The lipidoid formulations can include particles comprising either 3 or 4or more components in addition to polynucleotides. Lipidoids andpolynucleotide formulations comprising lipidoids are described in Intl.Pub. No. WO 2015051214 (herein incorporated by reference in itsentirety.

c. Hyaluronidase

In some embodiments, the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) and hyaluronidase for injection (e.g.,intramuscular or subcutaneous injection). Hyaluronidase catalyzes thehydrolysis of hyaluronan, which is a constituent of the interstitialbarrier. Hyaluronidase lowers the viscosity of hyaluronan, therebyincreases tissue permeability (Frost, Expert Opin. Drug Deliv. (2007)4:427-440). Alternatively, the hyaluronidase can be used to increase thenumber of cells exposed to the polynucleotides administeredintramuscularly, or subcutaneously.

d. Nanoparticle Mimics

In some embodiments, the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) is encapsulated within and/or absorbed to ananoparticle mimic. A nanoparticle mimic can mimic the delivery functionorganisms or particles such as, but not limited to, pathogens, viruses,bacteria, fungus, parasites, prions and cells. As a non-limitingexample, the polynucleotides described herein can be encapsulated in anon-viron particle that can mimic the delivery function of a virus (seee.g., Intl. Pub. No. WO2012006376 and U.S. Pub. Nos. US20130171241 andUS20130195968, each of which is herein incorporated by reference in itsentirety).

e. Self-Assembled Nanoparticles, or Self-Assembled Macromolecules

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) in self-assembled nanoparticles, or amphiphilicmacromolecules (AMs) for delivery. AMs comprise biocompatibleamphiphilic polymers that have an alkylated sugar backbone covalentlylinked to poly(ethylene glycol). In aqueous solution, the AMsself-assemble to form micelles. Nucleic acid self-assemblednanoparticles are described in Intl. Appl. No. PCT/US2014/027077, andAMs and methods of forming AMs are described in U.S. Pub. No.US20130217753, each of which is herein incorporated by reference in itsentirety.

f. Cations and Anions

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) and a cation or anion, such as Zn2+, Ca2+, Cu2+,Mg2+ and combinations thereof. Exemplary formulations can includepolymers and a polynucleotide complexed with a metal cation as describedin, e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which isherein incorporated by reference in its entirety. In some embodiments,cationic nanoparticles can contain a combination of divalent andmonovalent cations. The delivery of polynucleotides in cationicnanoparticles or in one or more depot comprising cationic nanoparticlescan improve polynucleotide bioavailability by acting as a long-actingdepot and/or reducing the rate of degradation by nucleases.

g. Amino Acid Lipids

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) that is formulation with an amino acid lipid.Amino acid lipids are lipophilic compounds comprising an amino acidresidue and one or more lipophilic tails. Non-limiting examples of aminoacid lipids and methods of making amino acid lipids are described inU.S. Pat. No. 8,501,824. The amino acid lipid formulations can deliver apolynucleotide in releasable form that comprises an amino acid lipidthat binds and releases the polynucleotides. As a non-limiting example,the release of the polynucleotides described herein can be provided byan acid-labile linker as described in, e.g., U.S. Pat. Nos. 7,098,032,6,897,196, 6,426,086, 7,138,382, 5,563,250, and 5,505,931, each of whichis herein incorporated by reference in its entirety.

h. Interpolyelectrolyte Complexes

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) in an interpolyelectrolyte complex.Interpolyelectrolyte complexes are formed when charge-dynamic polymersare complexed with one or more anionic molecules. Non-limiting examplesof charge-dynamic polymers and interpolyelectrolyte complexes andmethods of making interpolyelectrolyte complexes are described in U.S.Pat. No. 8,524,368, herein incorporated by reference in its entirety.

i. Crystalline Polymeric Systems

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) in crystalline polymeric systems. Crystallinepolymeric systems are polymers with crystalline moieties and/or terminalunits comprising crystalline moieties. Exemplary polymers are describedin U.S. Pat. No. 8,524,259 (herein incorporated by reference in itsentirety).

j. Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) and a natural and/or synthetic polymer. Thepolymers include, but not limited to, polyethenes, polyethylene glycol(PEG), poly(l-lysine)(PLL), PEG grafted to PLL, cationic lipopolymer,biodegradable cationic lipopolymer, polyethyleneimine (PEI),cross-linked branched poly(alkylene imines), a polyamine derivative, amodified poloxamer, elastic biodegradable polymer, biodegradablecopolymer, biodegradable polyester copolymer, biodegradable polyestercopolymer, multiblock copolymers, poly[α-(4-aminobutyl)-L-glycolic acid)(PAGA), biodegradable cross-linked cationic multi-block copolymers,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyureas, polystyrenes, polyamines,polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),amine-containing polymers, dextran polymers, dextran polymer derivativesor combinations thereof.

Exemplary polymers include, DYNAMIC POLYCONJUGATE® (Arrowhead ResearchCorp., Pasadena, CA) formulations from MIRUS® Bio (Madison, WI) andRoche Madison (Madison, WI), PHASERX™ polymer formulations such as,without limitation, SMARTT POLYMER TECHNOLOGY™ (PHASERX®, Seattle, WA),DMRI/DOPE, poloxamer, VAXFECTIN® adjuvant from Vical (San Diego, CA),chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA),dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers. RONDEL™(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (ArrowheadResearch Corporation, Pasadena, CA) and pH responsive co-block polymerssuch as PHASERX® (Seattle, WA).

The polymer formulations allow a sustained or delayed release of thepolynucleotide (e.g., following intramuscular or subcutaneousinjection). The altered release profile for the polynucleotide canresult in, for example, translation of an encoded protein over anextended period of time. The polymer formulation can also be used toincrease the stability of the polynucleotide. Sustained releaseformulations can include, but are not limited to, PLGA microspheres,ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics,Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA),surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia,GA), TISSELL® (Baxter International, Inc. Deerfield, IL), PEG-basedsealants, and COSEAL® (Baxter International, Inc. Deerfield, IL).

As a non-limiting example modified mRNA can be formulated in PLGAmicrospheres by preparing the PLGA microspheres with tunable releaserates (e.g., days and weeks) and encapsulating the modified mRNA in thePLGA microspheres while maintaining the integrity of the modified mRNAduring the encapsulation process. EVAc are non-biodegradable,biocompatible polymers that are used extensively in pre-clinicalsustained release implant applications (e.g., extended release productsOcusert a pilocarpine ophthalmic insert for glaucoma or progestasert asustained release progesterone intrauterine device; transdermal deliverysystems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407NF is a hydrophilic, non-ionic surfactant triblock copolymer ofpolyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosityat temperatures less than 5° C. and forms a solid gel at temperaturesgreater than 15° C.

As a non-limiting example, the polynucleotides described herein can beformulated with the polymeric compound of PEG grafted with PLL asdescribed in U.S. Pat. No. 6,177,274. As another non-limiting example,the polynucleotides described herein can be formulated with a blockcopolymer such as a PLGA-PEG block copolymer (see e.g., U.S. Pub. No.US20120004293 and U.S. Pat. Nos. 8,236,330 and 8,246,968), or aPLGA-PEG-PLGA block copolymer (see e.g., U.S. Pat. No. 6,004,573). Eachof the references is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated with at least one amine-containing polymer such as, but notlimited to polylysine, polyethylene imine, poly(amidoamine) dendrimers,poly(amine-co-esters) or combinations thereof. Exemplary polyaminepolymers and their use as delivery agents are described in, e.g., U.S.Pat. Nos. 8,460,696, 8,236,280, each of which is herein incorporated byreference in its entirety.

In some embodiments, the polynucleotides described herein can beformulated in a biodegradable cationic lipopolymer, a biodegradablepolymer, or a biodegradable copolymer, a biodegradable polyestercopolymer, a biodegradable polyester polymer, a linear biodegradablecopolymer, PAGA, a biodegradable cross-linked cationic multi-blockcopolymer or combinations thereof as described in, e.g., U.S. Pat. Nos.6,696,038, 6,517,869, 6,267,987, 6,217,912, 6,652,886, 8,057,821, and8,444,992; U.S. Pub. Nos. US20030073619, US20040142474, US20100004315,US2012009145 and US20130195920; and Intl Pub. Nos. WO2006063249 andW2013086322, each of which is herein incorporated by reference in itsentirety.

In some embodiments, the polynucleotides described herein can beformulated in or with at least one cyclodextrin polymer as described inU.S. Pub. No. US20130184453. In some embodiments, the polynucleotidesdescribed herein can be formulated in or with at least one crosslinkedcation-binding polymers as described in Intl. Pub. Nos. WO2013106072,WO2013106073 and WO2013106086. In some embodiments, the polynucleotidesdescribed herein can be formulated in or with at least PEGylated albuminpolymer as described in U.S. Pub. No. US20130231287. Each of thereferences is herein incorporated by reference in its entirety.

In some embodiments, the polynucleotides disclosed herein can beformulated as a nanoparticle using a combination of polymers, lipids,and/or other biodegradable agents, such as, but not limited to, calciumphosphate. Components can be combined in a core-shell, hybrid, and/orlayer-by-layer architecture, to allow for fine-tuning of thenanoparticle for delivery (Wang et al., Nat Mater. 2006 5:791-796;Fuller et al., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv DrugDeliv Rev. 2011 63:748-761; Endres et al., Biomaterials. 201132:7721-7731; Su et al., Mol Pharm. 2011 Jun. 6; 8(3):774-87; hereinincorporated by reference in their entireties). As a non-limitingexample, the nanoparticle can comprise a plurality of polymers such as,but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA),hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (Intl. Pub.No. WO20120225129, herein incorporated by reference in its entirety).

The use of core-shell nanoparticles has additionally focused on ahigh-throughput approach to synthesize cationic cross-linked nanogelcores and various shells (Siegwart et al., Proc Natl Acad Sci USA. 2011108:12996-13001; herein incorporated by reference in its entirety). Thecomplexation, delivery, and internalization of the polymericnanoparticles can be precisely controlled by altering the chemicalcomposition in both the core and shell components of the nanoparticle.For example, the core-shell nanoparticles can efficiently deliver siRNAto mouse hepatocytes after they covalently attach cholesterol to thenanoparticle.

In some embodiments, a hollow lipid core comprising a middle PLGA layerand an outer neutral lipid layer containing PEG can be used to deliveryof the polynucleotides as described herein. In some embodiments, thelipid nanoparticles can comprise a core of the polynucleotides disclosedherein and a polymer shell, which is used to protect the polynucleotidesin the core. The polymer shell can be any of the polymers describedherein and are known in the art. The polymer shell can be used toprotect the polynucleotides in the core.

Core-shell nanoparticles for use with the polynucleotides describedherein are described in U.S. Pat. No. 8,313,777 or Intl. Pub. No.WO2013124867, each of which is herein incorporated by reference in theirentirety.

k. Peptides and Proteins

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) that is formulated with peptides and/or proteinsto increase transfection of cells by the polynucleotide, and/or to alterthe biodistribution of the polynucleotide (e.g., by targeting specifictissues or cell types), and/or increase the translation of encodedprotein (e.g., Intl. Pub. Nos. WO2012110636 and WO2013123298. In someembodiments, the peptides can be those described in U.S. Pub. Nos.US20130129726, US20130137644 and US20130164219. Each of the referencesis herein incorporated by reference in its entirety.

l. Conjugates

In some embodiments, the compositions or formulations of the presentdisclosure comprise the polynucleotides described herein (e.g., apolynucleotide comprising a nucleotide sequence encoding an anti-CHIKVantibody polypeptide) that is covalently linked to a carrier ortargeting group, or including two encoding regions that together producea fusion protein (e.g., bearing a targeting group and therapeuticprotein or peptide) as a conjugate. The conjugate can be a peptide thatselectively directs the nanoparticle to neurons in a tissue or organism,or assists in crossing the blood-brain barrier.

The conjugates include a naturally occurring substance, such as aprotein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand can also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g., an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

In some embodiments, the conjugate can function as a carrier for thepolynucleotide disclosed herein. The conjugate can comprise a cationicpolymer such as, but not limited to, polyamine, polylysine,polyalkylenimine, and polyethylenimine that can be grafted to withpoly(ethylene glycol). Exemplary conjugates and their preparations aredescribed in U.S. Pat. No. 6,586,524 and U.S. Pub. No. US20130211249,each of which herein is incorporated by reference in its entirety.

The conjugates can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer.

Targeting groups can be proteins, e.g., glycoproteins, or peptides,e.g., molecules having a specific affinity for a co-ligand, orantibodies e.g., an antibody, that binds to a specified cell type suchas an endothelial cell or bone cell. Targeting groups can also includehormones and hormone receptors. They can also include non-peptidicspecies, such as lipids, lectins, carbohydrates, vitamins, cofactors,multivalent lactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-glucosamine multivalent mannose, multivalent frucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, or anactivator of p38 MAP kinase.

The targeting group can be any ligand that is capable of targeting aspecific receptor. Examples include, without limitation, folate, GaNAc,galactose, mannose, mannose-6P, apatamers, integrin receptor ligands,chemokine receptor ligands, transferrin, biotin, serotonin receptorligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. Inparticular embodiments, the targeting group is an aptamer. The aptamercan be unmodified or have any combination of modifications disclosedherein. As a non-limiting example, the targeting group can be aglutathione receptor (GR)-binding conjugate for targeted delivery acrossthe blood-central nervous system barrier as described in, e.g., U.S.Pub. No. US2013021661012 (herein incorporated by reference in itsentirety).

In some embodiments, the conjugate can be a synergisticbiomolecule-polymer conjugate, which comprises a long-actingcontinuous-release system to provide a greater therapeutic efficacy. Thesynergistic biomolecule-polymer conjugate can be those described in U.S.Pub. No. US20130195799. In some embodiments, the conjugate can be anaptamer conjugate as described in Intl. Pat. Pub. No. WO2012040524. Insome embodiments, the conjugate can be an amine containing polymerconjugate as described in U.S. Pat. No. 8,507,653. Each of thereferences is herein incorporated by reference in its entirety. In someembodiments, the polynucleotides can be conjugated to SMARTT POLYMERTECHNOLOGY® (PHASERX®, Inc. Seattle, WA).

In some embodiments, the polynucleotides described herein are covalentlyconjugated to a cell penetrating polypeptide, which can also include asignal sequence or a targeting sequence. The conjugates can be designedto have increased stability, and/or increased cell transfection; and/oraltered the biodistribution (e.g., targeted to specific tissues or celltypes).

In some embodiments, the polynucleotides described herein can beconjugated to an agent to enhance delivery. In some embodiments, theagent can be a monomer or polymer such as a targeting monomer or apolymer having targeting blocks as described in Intl. Pub. No.WO2011062965. In some embodiments, the agent can be a transport agentcovalently coupled to a polynucleotide as described in, e.g., U.S. Pat.Nos. 6,835,393 and 7,374,778. In some embodiments, the agent can be amembrane barrier transport enhancing agent such as those described inU.S. Pat. Nos. 7,737,108 and 8,003,129. Each of the references is hereinincorporated by reference in its entirety.

22. Accelerated Blood Clearance

The invention provides compounds, compositions and methods of usethereof for reducing the effect of ABC on a repeatedly administeredactive agent such as a biologically active agent. As will be readilyapparent, reducing or eliminating altogether the effect of ABC on anadministered active agent effectively increases its half-life and thusits efficacy.

In some embodiments the term reducing ABC refers to any reduction in ABCin comparison to a positive reference control ABC inducing LNP such asan MC3 LNP. ABC inducing LNPs cause a reduction in circulating levels ofan active agent upon a second or subsequent administration within agiven time frame. Thus a reduction in ABC refers to less clearance ofcirculating agent upon a second or subsequent dose of agent, relative toa standard LNP. The reduction may be, for instance, at least 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 100%. In some embodiments the reduction is 10-100%,10-50%, 20-100%, 20-50%, 30-100%, 30-50%, 40%-100%, 40-80%, 50-90%, or50-100%. Alternatively, the reduction in ABC may be characterized as atleast a detectable level of circulating agent following a second orsubsequent administration or at least a 2 fold, 3 fold, 4 fold, 5 foldincrease in circulating agent relative to circulating agent followingadministration of a standard LNP. In some embodiments the reduction is a2-100 fold, 2-50 fold, 3-100 fold, 3-50 fold, 3-20 fold, 4-100 fold,4-50 fold, 4-40 fold, 4-30 fold, 4-25 fold, 4-20 fold, 4-15 fold, 4-10fold, 4-5 fold, 5-100 fold, 5-50 fold, 5-40 fold, 5-30 fold, 5-25 fold,5-20 fold, 5-15 fold, 5-10 fold, 6-100 fold, 6-50 fold, 6-40 fold, 6-30fold, 6-25 fold, 6-20 fold, 6-15 fold, 6-10 fold, 8-100 fold, 8-50 fold,8-40 fold, 8-30 fold, 8-25 fold, 8-20 fold, 8-15 fold, 8-10 fold, 10-100fold, 10-50 fold, 10-40 fold, 10-30 fold, 10-25 fold, 10-20 fold, 10-15fold, 20-100 fold, 20-50 fold, 20-fold, 20-30 fold, or 20-25 fold.

The disclosure provides lipid-comprising compounds and compositions thatare less susceptible to clearance and thus have a longer half-life invivo. This is particularly the case where the compositions are intendedfor repeated including chronic administration, and even moreparticularly where such repeated administration occurs within days orweeks.

Significantly, these compositions are less susceptible or altogethercircumvent the observed phenomenon of accelerated blood clearance (ABC).ABC is a phenomenon in which certain exogenously administered agents arerapidly cleared from the blood upon second and subsequentadministrations. This phenomenon has been observed, in part, for avariety of lipid-containing compositions including but not limited tolipidated agents, liposomes or other lipid-based delivery vehicles, andlipid-encapsulated agents. Heretofore, the basis of ABC has been poorlyunderstood and in some cases attributed to a humoral immune response andaccordingly strategies for limiting its impact in vivo particularly in aclinical setting have remained elusive.

This disclosure provides compounds and compositions that are lesssusceptible, if at all susceptible, to ABC. In some important aspects,such compounds and compositions are lipid-comprising compounds orcompositions. The lipid-containing compounds or compositions of thisdisclosure, surprisingly, do not experience ABC upon second andsubsequent administration in vivo. This resistance to ABC renders thesecompounds and compositions particularly suitable for repeated use invivo, including for repeated use within short periods of time, includingdays or 1-2 weeks. This enhanced stability and/or half-life is due, inpart, to the inability of these compositions to activate B1a and/or B1bcells and/or conventional B cells, pDCs and/or platelets.

This disclosure therefore provides an elucidation of the mechanismunderlying accelerated blood clearance (ABC). It has been found, inaccordance with this disclosure and the inventions provided herein, thatthe ABC phenomenon at least as it relates to lipids and lipidnanoparticles is mediated, at least in part an innate immune responseinvolving B1a and/or B1b cells, pDC and/or platelets. B1a cells arenormally responsible for secreting natural antibody, in the form ofcirculating IgM. This IgM is poly-reactive, meaning that it is able tobind to a variety of antigens, albeit with a relatively low affinity foreach.

It has been found in accordance with the invention that some lipidatedagents or lipid-comprising formulations such as lipid nanoparticlesadministered in vivo trigger and are subject to ABC. It has now beenfound in accordance with the invention that upon administration of afirst dose of the LNP, one or more cells involved in generating aninnate immune response (referred to herein as sensors) bind such agent,are activated, and then initiate a cascade of immune factors (referredto herein as effectors) that promote ABC and toxicity. For instance, B1aand B1b cells may bind to LNP, become activated (alone or in thepresence of other sensors such as pDC and/or effectors such as IL6) andsecrete natural IgM that binds to the LNP. Pre-existing natural IgM inthe subject may also recognize and bind to the LNP, thereby triggeringcomplement fixation. After administration of the first dose, theproduction of natural IgM begins within 1-2 hours of administration ofthe LNP. Typically, by about 2-3 weeks the natural IgM is cleared fromthe system due to the natural half-life of IgM. Natural IgG is producedbeginning around 96 hours after administration of the LNP. The agent,when administered in a naïve setting, can exert its biological effectsrelatively unencumbered by the natural IgM produced post-activation ofthe B1a cells or B1b cells or natural IgG. The natural IgM and naturalIgG are non-specific and thus are distinct from anti-PEG IgM andanti-PEG IgG.

Although Applicant is not bound by mechanism, it is proposed that LNPstrigger ABC and/or toxicity through the following mechanisms. It isbelieved that when an LNP is administered to a subject the LNP israpidly transported through the blood to the spleen. The LNPs mayencounter immune cells in the blood and/or the spleen. A rapid innateimmune response is triggered in response to the presence of the LNPwithin the blood and/or spleen. Applicant has shown herein that withinhours of administration of an LNP several immune sensors have reacted tothe presence of the LNP. These sensors include but are not limited toimmune cells involved in generating an immune response, such as B cells,pDC, and platelets. The sensors may be present in the spleen, such as inthe marginal zone of the spleen and/or in the blood. The LNP mayphysically interact with one or more sensors, which may interact withother sensors. In such a case the LNP is directly or indirectlyinteracting with the sensors. The sensors may interact directly with oneanother in response to recognition of the LNP. For instance, manysensors are located in the spleen and can easily interact with oneanother. Alternatively, one or more of the sensors may interact with LNPin the blood and become activated. The activated sensor may theninteract directly with other sensors or indirectly (e.g., through thestimulation or production of a messenger such as a cytokine e.g., IL6).

In some embodiments the LNP may interact directly with and activate eachof the following sensors: pDC, B1a cells, B1b cells, and platelets.These cells may then interact directly or indirectly with one another toinitiate the production of effectors which ultimately lead to the ABCand/or toxicity associated with repeated doses of LNP. For instance,Applicant has shown that LNP administration leads to pDC activation,platelet aggregation and activation and B cell activation. In responseto LNP platelets also aggregate and are activated and aggregate with Bcells. pDC cells are activated. LNP has been found to interact with thesurface of platelets and B cells relatively quickly. Blocking theactivation of any one or combination of these sensors in response to LNPis useful for dampening the immune response that would ordinarily occur.This dampening of the immune response results in the avoidance of ABCand/or toxicity.

The sensors once activated produce effectors. An effector, as usedherein, is an immune molecule produced by an immune cell, such as a Bcell. Effectors include but are not limited to immunoglobulin such asnatural IgM and natural IgG and cytokines such as IL6. B1a and B1b cellsstimulate the production of natural IgMs within 2-6 hours followingadministration of an LNP. Natural IgG can be detected within 96 hours.IL6 levels are increased within several hours. The natural IgM and IgGcirculate in the body for several days to several weeks. During thistime the circulating effectors can interact with newly administeredLNPs, triggering those LNPs for clearance by the body. For instance, aneffector may recognize and bind to an LNP. The Fc region of the effectormay be recognized by and trigger uptake of the decorated LNP bymacrophage. The macrophage are then transported to the spleen. Theproduction of effectors by immune sensors is a transient response thatcorrelates with the timing observed for ABC.

If the administered dose is the second or subsequent administered dose,and if such second or subsequent dose is administered before thepreviously induced natural IgM and/or IgG is cleared from the system(e.g., before the 2-3 window time period), then such second orsubsequent dose is targeted by the circulating natural IgM and/ornatural IgG or Fc which trigger alternative complement pathwayactivation and is itself rapidly cleared. When LNP are administeredafter the effectors have cleared from the body or are reduced in number,ABC is not observed.

Thus, it is useful according to aspects of the invention to inhibit theinteraction between LNP and one or more sensors, to inhibit theactivation of one or more sensors by LNP (direct or indirect), toinhibit the production of one or more effectors, and/or to inhibit theactivity of one or more effectors. In some embodiments the LNP isdesigned to limit or block interaction of the LNP with a sensor. Forinstance, the LNP may have an altered PC and/or PEG to preventinteractions with sensors. Alternatively, or additionally, an agent thatinhibits immune responses induced by LNPs may be used to achieve any oneor more of these effects.

It has also been determined that conventional B cells are alsoimplicated in ABC. Specifically, upon first administration of an agent,conventional B cells, referred to herein as CD19(+), bind to and reactagainst the agent. Unlike B1a and Bb cells though, conventional B cellsare able to mount first an IgM response (beginning around 96 hours afteradministration of the LNPs) followed by an IgG response (beginningaround 14 days after administration of the LNPs) concomitant with amemory response. Thus conventional B cells react against theadministered agent and contribute to IgM (and eventually IgG) thatmediates ABC. The IgM and IgG are typically anti-PEG IgM and anti-PEGIgG.

It is contemplated that in some instances, the majority of the ABCresponse is mediated through B1a cells and B1a-mediated immuneresponses. It is further contemplated that in some instances, the ABCresponse is mediated by both IgM and IgG, with both conventional B cellsand B1a cells mediating such effects. In yet still other instances, theABC response is mediated by natural IgM molecules, some of which arecapable of binding to natural IgM, which may be produced by activatedB1a cells. The natural IgMs may bind to one or more components of theLNPs, e.g., binding to a phospholipid component of the LNPs (such asbinding to the PC moiety of the phospholipid) and/or binding to aPEG-lipid component of the LNPs (such as binding to PEG-DMG, inparticular, binding to the PEG moiety of PEG-DMG). Since B1a expressesCD36, to which phosphatidylcholine is a ligand, it is contemplated thatthe CD36 receptor may mediate the activation of B1a cells and thusproduction of natural IgM. In yet still other instances, the ABCresponse is mediated primarily by conventional B cells.

It has been found in accordance with the invention that the ABCphenomenon can be reduced or abrogated, at least in part, through theuse of compounds and compositions (such as agents, delivery vehicles,and formulations) that do not activate B1a cells. Compounds andcompositions that do not activate B1a cells may be referred to herein asB1a inert compounds and compositions. It has been further found inaccordance with the invention that the ABC phenomenon can be reduced orabrogated, at least in part, through the use of compounds andcompositions that do not activate conventional B cells. Compounds andcompositions that do not activate conventional B cells may in someembodiments be referred to herein as CD19-inert compounds andcompositions. Thus, in some embodiments provided herein, the compoundsand compositions do not activate B1a cells and they do not activateconventional B cells. Compounds and compositions that do not activateB1a cells and conventional B cells may in some embodiments be referredto herein as B1a/CD19-inert compounds and compositions.

These underlying mechanisms were not heretofore understood, and the roleof B1a and B1b cells and their interplay with conventional B cells inthis phenomenon was also not appreciated.

Accordingly, this disclosure provides compounds and compositions that donot promote ABC. These may be further characterized as not capable ofactivating B1a and/or Bib cells, platelets and/or pDC, and optionallyconventional B cells also. These compounds (e.g., agents, includingbiologically active agents such as prophylactic agents, therapeuticagents and diagnostic agents, delivery vehicles, including liposomes,lipid nanoparticles, and other lipid-based encapsulating structures,etc.) and compositions (e.g., formulations, etc.) are particularlydesirable for applications requiring repeated administration, and inparticular repeated administrations that occur within with short periodsof time (e.g., within 1-2 weeks). This is the case, for example, if theagent is a nucleic acid based therapeutic that is provided to a subjectat regular, closely-spaced intervals. The findings provided herein maybe applied to these and other agents that are similarly administeredand/or that are subject to ABC.

Of particular interest are lipid-comprising compounds, lipid-comprisingparticles, and lipid-comprising compositions as these are known to besusceptible to ABC. Such lipid-comprising compounds particles, andcompositions have been used extensively as biologically active agents oras delivery vehicles for such agents. Thus, the ability to improve theirefficacy of such agents, whether by reducing the effect of ABC on theagent itself or on its delivery vehicle, is beneficial for a widevariety of active agents.

Also provided herein are compositions that do not stimulate or boost anacute phase response (ARP) associated with repeat dose administration ofone or more biologically active agents.

The composition, in some instances, may not bind to IgM, including butnot limited to natural IgM.

The composition, in some instances, may not bind to an acute phaseprotein such as but not limited to C-reactive protein.

The composition, in some instances, may not trigger a CD5(+) mediatedimmune response. As used herein, a CD5(+) mediated immune response is animmune response that is mediated by B1a and/or B1b cells. Such aresponse may include an ABC response, an acute phase response, inductionof natural IgM and/or IgG, and the like.

The composition, in some instances, may not trigger a CD19(+) mediatedimmune response. As used herein, a CD19(+) mediated immune response isan immune response that is mediated by conventional CD19(+), CD5(−) Bcells. Such a response may include induction of IgM, induction of IgG,induction of memory B cells, an ABC response, an anti-drug antibody(ADA) response including an anti-protein response where the protein maybe encapsulated within an LNP, and the like.

B1a cells area subset of B cells involved in innate immunity. Thesecells are the source of circulating IgM, referred to as natural antibodyor natural serum antibody. Natural IgM antibodies are characterized ashaving weak affinity for a number of antigens, and therefore they arereferred to as “poly-specific” or “poly-reactive”, indicating theirability to bind to more than one antigen. B1a cells are not able toproduce IgG. Additionally, they do not develop into memory cells andthus do not contribute to an adaptive immune response. However, they areable to secrete IgM upon activation. The secreted IgM is typicallycleared within about 2-3 weeks, at which point the immune system isrendered relatively naïve to the previously administered antigen. If thesame antigen is presented after this time period (e.g., at about 3 weeksafter the initial exposure), the antigen is not rapidly cleared.However, significantly, if the antigen is presented within that timeperiod (e.g., within 2 weeks, including within 1 week, or within days),then the antigen is rapidly cleared. This delay between consecutivedoses has rendered certain lipid-containing therapeutic or diagnosticagents unsuitable for use.

In humans, B1a cells are CD19(+), CD20(+), CD27(+), CD43(+), CD70(−) andCD5(+). In mice, B1a cells are CD19(+), CD5(+), and CD45 B cell isoformB220(+). It is the expression of CD5 which typically distinguishes B1acells from other convention B cells. B1a cells may express high levelsof CD5, and on this basis may be distinguished from other B-1 cells suchas B-1b cells which express low or undetectable levels of CD5. CD5 is apan-T cell surface glycoprotein. B1a cells also express CD36, also knownas fatty acid translocase. CD36 is a member of the class B scavengerreceptor family. CD36 can bind many ligands, including oxidized lowdensity lipoproteins, native lipoproteins, oxidized phospholipids, andlong-chain fatty acids.

B1b cells are another subset of B cells involved in innate immunity.These cells are another source of circulating natural IgM. Severalantigens, including PS, are capable of inducing T cell independentimmunity through B1b activation. CD27 is typically upregulated on B1bcells in response to antigen activation. Similar to B1a cells, the Bbcells are typically located in specific body locations such as thespleen and peritoneal cavity and are in very low abundance in the blood.The B1b secreted natural IgM is typically cleared within about 2-3weeks, at which point the immune system is rendered relatively naïve tothe previously administered antigen. If the same antigen is presentedafter this time period (e.g., at about 3 weeks after the initialexposure), the antigen is not rapidly cleared. However, significantly,if the antigen is presented within that time period (e.g., within 2weeks, including within 1 week, or within days), then the antigen israpidly cleared. This delay between consecutive doses has renderedcertain lipid-containing therapeutic or diagnostic agents unsuitable foruse.

In some embodiments it is desirable to block B1a and/or B1b cellactivation. One strategy for blocking B1a and/or B1b cell activationinvolves determining which components of a lipid nanoparticle promote Bcell activation and neutralizing those components. It has beendiscovered herein that at least PEG and phosphatidylcholine (PC)contribute to B1a and B1b cell interaction with other cells and/oractivation. PEG may play a role in promoting aggregation between B1cells and platelets, which may lead to activation. PC (a helper lipid inLNPs) is also involved in activating the B1 cells, likely throughinteraction with the CD36 receptor on the B cell surface. Numerousparticles have PEG-lipid alternatives, PEG-less, and/or PC replacementlipids (e.g. oleic acid or analogs thereof) have been designed andtested. Applicant has established that replacement of one or more ofthese components within an LNP that otherwise would promote ABC uponrepeat administration, is useful in preventing ABC by reducing theproduction of natural IgM and/or B cell activation. Thus, the inventionencompasses LNPs that have reduced ABC as a result of a design whicheliminates the inclusion of B cell triggers.

Another strategy for blocking B1a and/or B1b cell activation involvesusing an agent that inhibits immune responses induced by LNPs. Thesetypes of agents are discussed in more detail below. In some embodimentsthese agents block the interaction between B1a/B1b cells and the LNP orplatelets or pDC. For instance, the agent may be an antibody or otherbinding agent that physically blocks the interaction. An example of thisis an antibody that binds to CD36 or CD6. The agent may also be acompound that prevents or disables the B1a/B1b cell from signaling onceactivated or prior to activation. For instance, it is possible to blockone or more components in the B1a/B1b signaling cascade the results fromB cell interaction with LNP or other immune cells. In other embodimentsthe agent may act one or more effectors produced by the B1a/B1b cellsfollowing activation. These effectors include for instance, natural IgMand cytokines.

It has been demonstrated according to aspects of the invention that whenactivation of pDC cells is blocked, B cell activation in response to LNPis decreased. Thus, in order to avoid ABC and/or toxicity, it may bedesirable to prevent pDC activation. Similar to the strategies discussedabove, pDC cell activation may be blocked by agents that interfere withthe interaction between pDC and LNP and/or B cells/platelets.Alternatively, agents that act on the pDC to block its ability to getactivated or on its effectors can be used together with the LNP to avoidABC.

Platelets may also play an important role in ABC and toxicity. Veryquickly after a first dose of LNP is administered to a subject plateletsassociate with the LNP, aggregate and are activated. In some embodimentsit is desirable to block platelet aggregation and/or activation. Onestrategy for blocking platelet aggregation and/or activation involvesdetermining which components of a lipid nanoparticle promote plateletaggregation and/or activation and neutralizing those components. It hasbeen discovered herein that at least PEG contribute to plateletaggregation, activation and/or interaction with other cells. Numerousparticles have PEG-lipid alternatives and PEG-less have been designedand tested. Applicant has established that replacement of one or more ofthese components within an LNP that otherwise would promote ABC uponrepeat administration, is useful in preventing ABC by reducing theproduction of natural IgM and/or platelet aggregation. Thus, theinvention encompasses LNPs that have reduced ABC as a result of a designwhich eliminates the inclusion of platelet triggers. Alternativelyagents that act on the platelets to block its activity once it isactivated or on its effectors can be used together with the LNP to avoidABC.

(i) Measuring ABC Activity and Related Activities Various compounds andcompositions provided herein, including LNPs, do not promote ABCactivity upon administration in vivo. These LNPs may be characterizedand/or identified through any of a number of assays, such as but notlimited to those described below, as well as any of the assays disclosedin the Examples section, include the methods subsection of the Examples.

In some embodiments the methods involve administering an LNP withoutproducing an immune response that promotes ABC. An immune response thatpromotes ABC involves activation of one or more sensors, such as B1cells, pDC, or platelets, and one or more effectors, such as naturalIgM, natural IgG or cytokines such as IL6. Thus administration of an LNPwithout producing an immune response that promotes ABC, at a minimuminvolves administration of an LNP without significant activation of oneor more sensors and significant production of one or more effectors.Significant used in this context refers to an amount that would lead tothe physiological consequence of accelerated blood clearance of all orpart of a second dose with respect to the level of blood clearanceexpected for a second dose of an ABC triggering LNP. For instance, theimmune response should be dampened such that the ABC observed after thesecond dose is lower than would have been expected for an ABC triggeringLNP.

(ii) B1a or B1b Activation Assay

Certain compositions provided in this disclosure do not activate Bcells, such as B1a or B1b cells (CD19+ CD5+) and/or conventional B cells(CD19+ CD5−). Activation of B1a cells, B1b cells, or conventional Bcells may be determined in a number of ways, some of which are providedbelow. B cell population may be provided as fractionated B cellpopulations or unfractionated populations of splenocytes or peripheralblood mononuclear cells (PBMC). If the latter, the cell population maybe incubated with the LNP of choice for a period of time, and thenharvested for further analysis. Alternatively, the supernatant may beharvested and analyzed.

(iii) Upregulation of Activation Marker Cell Surface Expression

Activation of B1a cells, B1b cells, or conventional B cells may bedemonstrated as increased expression of B cell activation markersincluding late activation markers such as CD86. In an exemplarynon-limiting assay, unfractionated B cells are provided as a splenocytepopulation or as a PBMC population, incubated with an LNP of choice fora particular period of time, and then stained for a standard B cellmarker such as CD19 and for an activation marker such as CD86, andanalyzed using for example flow cytometry. A suitable negative controlinvolves incubating the same population with medium, and then performingthe same staining and visualization steps. An increase in CD86expression in the test population compared to the negative controlindicates B cell activation.

(iv) Pro-Inflammatory Cytokine Release

B cell activation may also be assessed by cytokine release assay. Forexample, activation may be assessed through the production and/orsecretion of cytokines such as IL-6 and/or TNF-alpha upon exposure withLNPs of interest.

Such assays may be performed using routine cytokine secretion assayswell known in the art. An increase in cytokine secretion is indicativeof B cell activation.

(v) LNP Binding/Association to and/or Uptake by B Cells

LNP association or binding to B cells may also be used to assess an LNPof interest and to further characterize such LNP. Association/bindingand/or uptake/internalization may be assessed using a detectablylabeled, such as fluorescently labeled, LNP and tracking the location ofsuch LNP in or on B cells following various periods of incubation.

The invention further contemplates that the compositions provided hereinmay be capable of evading recognition or detection and optionallybinding by downstream mediators of ABC such as circulating IgM and/oracute phase response mediators such as acute phase proteins (e.g.,C-reactive protein (CRP).

(vi) Methods of Use for Reducing ABC

Also provided herein are methods for delivering LNPs, which mayencapsulate an agent such as a therapeutic agent, to a subject withoutpromoting ABC.

In some embodiments, the method comprises administering any of the LNPsdescribed herein, which do not promote ABC, for example, do not induceproduction of natural IgM binding to the LNPs, do not activate B1aand/or B1b cells. As used herein, an LNP that “does not promote ABC”refers to an LNP that induces no immune responses that would lead tosubstantial ABC or a substantially low level of immune responses that isnot sufficient to lead to substantial ABC. An LNP that does not inducethe production of natural IgMs binding to the LNP refers to LNPs thatinduce either no natural IgM binding to the LNPs or a substantially lowlevel of the natural IgM molecules, which is insufficient to lead tosubstantial ABC. An LNP that does not activate B1a and/or B1b cellsrefer to LNPs that induce no response of B1a and/or B1b cells to producenatural IgM binding to the LNPs or a substantially low level of B1aand/or B1b responses, which is insufficient to lead to substantial ABC.

In some embodiments the terms do not activate and do not induceproduction are a relative reduction to a reference value or condition.In some embodiments the reference value or condition is the amount ofactivation or induction of production of a molecule such as IgM by astandard LNP such as an MC3 LNP. In some embodiments the relativereduction is a reduction of at least 30%, for example at least 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments the terms donot activate cells such as B cells and do not induce production of aprotein such as IgM may refer to an undetectable amount of the activecells or the specific protein.

(vii) Platelet Effects and Toxicity

The invention is further premised in part on the elucidation of themechanism underlying dose-limiting toxicity associated with LNPadministration. Such toxicity may involve coagulopathy, disseminatedintravascular coagulation (DIC, also referred to as consumptivecoagulopathy), whether acute or chronic, and/or vascular thrombosis. Insome instances, the dose-limiting toxicity associated with LNPs is acutephase response (APR) or complement activation-related psudoallergy(CARPA).

As used herein, coagulopathy refers to increased coagulation (bloodclotting) in vivo. The findings reported in this disclosure areconsistent with such increased coagulation and significantly provideinsight on the underlying mechanism. Coagulation is a process thatinvolves a number of different factors and cell types, and heretoforethe relationship between and interaction of LNPs and platelets has notbeen understood in this regard. This disclosure provides evidence ofsuch interaction and also provides compounds and compositions that aremodified to have reduced platelet effect, including reduced plateletassociation, reduced platelet aggregation, and/or reduced plateletaggregation. The ability to modulate, including preferablydown-modulate, such platelet effects can reduce the incidence and/orseverity of coagulopathy post-LNP administration. This in turn willreduce toxicity relating to such LNP, thereby allowing higher doses ofLNPs and importantly their cargo to be administered to patients in needthereof.

CARPA is a class of acute immune toxicity manifested in hypersensitivityreactions (HSRs), which may be triggered by nanomedicines andbiologicals. Unlike allergic reactions, CARPA typically does not involveIgE but arises as a consequence of activation of the complement system,which is part of the innate immune system that enhances the body'sabilities to clear pathogens. One or more of the following pathways, theclassical complement pathway (CP), the alternative pathway (AP), and thelectin pathway (LP), may be involved in CARPA. Szebeni, MolecularImmunology, 61:163-173 (2014).

The classical pathway is triggered by activation of the C1-complex,which contains. C1q, C1r, C1s, or C1qr2s2. Activation of the C1-complexoccurs when C1q binds to IgM or IgG complexed with antigens, or when C1q binds directly to the surface of the pathogen. Such binding leads toconformational changes in the C1 q molecule, which leads to theactivation of C1r, which in turn, cleave C1s. The C1r2s2 component nowsplits C4 and then C2, producing C4a, C4b, C2a, and C2b. C4b and C2bbind to form the classical pathway C3-convertase (C4b2b complex), whichpromotes cleavage of C3 into C3a and C3b. C3b then binds the C3convertase to from the C5 convertase (C4b2b3b complex). The alternativepathway is continuously activated as a result of spontaneous C3hydrolysis. Factor P (properdin) is a positive regulator of thealternative pathway. Oligomerization of properdin stabilizes the C3convertase, which can then cleave much more C3. The C3 molecules canbind to surfaces and recruit more B, D, and P activity, leading toamplification of the complement activation.

Acute phase response (APR) is a complex systemic innate immune responsefor preventing infection and clearing potential pathogens. Numerousproteins are involved in APR and C-reactive protein is awell-characterized one.

It has been found, in accordance with the invention, that certain LNPare able to associate physically with platelets almost immediately afteradministration in vivo, while other LNP do not associate with plateletsat all or only at background levels. Significantly, those LNPs thatassociate with platelets also apparently stabilize the plateletaggregates that are formed thereafter. Physical contact of the plateletswith certain LNPs correlates with the ability of such platelets toremain aggregated or to form aggregates continuously for an extendedperiod of time after administration. Such aggregates comprise activatedplatelets and also innate immune cells such as macrophages and B cells.

23. Methods of Use

The polynucleotides, pharmaceutical compositions and formulationsdescribed herein are used in the preparation, manufacture andtherapeutic use of to treat and/or prevent diseases, disorders orconditions related to chikungunya virus (CHIKV) infection. In someembodiments, the polynucleotides, compositions and formulations of thepresent disclosure are used to treat and/or prevent infectious diseasesuch as chikungunya fever.

In some embodiments, the polynucleotides, pharmaceutical compositionsand formulations of the invention (e.g., at least one mRNA encodinganti-CHIKV antibody polypeptide, such as mRNAs expressing the heavy andlight chains of anti-CHIKV antibody) are used in methods for reducingthe levels of virus in a subject in need thereof, e.g., a subjectinfected with chikungunya virus. In some embodiments, administration ofa polynucleotide, pharmaceutical composition, or formulation describedherein, to a subject reduces the viral load of CHIKV in the subject,e.g., reduces the viral load of CHIKV in the tissues and/or blood of asubject. In some embodiments, administration of a polynucleotide,pharmaceutical composition, or formulation described herein, to asubject reduces the amount of virus in the subject by at least 10%,e.g., by 10%, 15%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100%. In some embodiments, administration of apolynucleotide, pharmaceutical composition, or formulation describedherein, to a subject reduces the amount of virus in a particular tissueof the subject by at least 10%, e.g., by 10%, 15%, 20%, 30%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. In someembodiments, administration of a polynucleotide, pharmaceuticalcomposition, or formulation described herein, to a subject reducesviremia in a subject, e.g., by reducing the amount of virus in the bloodof a subject by at least 10%, e.g., by 10%, 15%, 20%, 30%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

One aspect of the invention provides a method of neutralizing CHIKVinfection in a subject comprising the administration of apolynucleotide, pharmaceutical composition, or formulation describedherein to a subject (e.g., at least one mRNA encoding anti-CHIKVantibody polypeptide, such as mRNAs expressing the heavy and lightchains of anti-CHIKV antibody). In some embodiments, administration of apolynucleotide, pharmaceutical composition, or formulation describedherein to a subject causes the expression of at least one anti-CHIKVantibody polynucleotide in the subject, wherein the at least oneanti-CHIKV antibody polynucleotide binds specifically to CHIKV andneutralizes CHIKV in the subject, thereby preventing or reducing thelevels of further virus infection. In some embodiments, mRNAs encodingthe heavy and light chains of anti-CHIKV antibody are administered to asubject infected with CHIKV, so that the heavy and light chains areexpressed in the subject and associate to form anti-CHIKV antibody thatneutralizes CHIKV in the subject. The anti-CHIKV antibody polypeptidesdescribed herein can neutralize CHIKV by several potential mechanisms,including, by way of example of example only, interfering with virionbinding to receptors, blocking uptake of virions into cells, preventinguncoating of virus genomes in endosomes, or causing aggregation of virusparticles. In some embodiments, administration of a polynucleotide,pharmaceutical composition, or formulation described herein neutralizesat least 10% of the CHIKV virions in a subject infected with CHIKV,e.g., neutralizes 10%, 15%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, or 100% of the CHIKV virions in a subject.

One aspect of the invention provides a method of alleviating thesymptoms of CHIKV infection in a subject that is known to be infectedwith CHIKV, or suspected of being infected with CHIKV, comprising theadministration of a polynucleotide, composition or formulation describedherein to a subject (e.g., at least one mRNA encoding anti-CHIKVantibody polypeptide, such as mRNAs expressing the heavy and lightchains of anti-CHIKV antibody). Chikungunya fever is an acute febrileillness that is caused by chikungunya virus infection. Chikungunya feversymptoms develop abruptly with high fever that can last for severaldays, and severe and often debilitating polyarthralgias. Arthritis withjoint swelling can also occur. In some cases, individuals infected withCHIKV can develop a maculopapular rash, and/or non-specific symptoms,such as headache, fatigue, nausea, vomiting, conjunctivitis, andmyalgias. In some embodiments, administration of a polynucleotide,pharmaceutical composition, or formulation described herein to a subjectwith chikungunya fever reduces the severity of at least one symptom ofthe disease in the subject, e.g., reduces the severity fever and/orpolyarthralgia in a subject. In some embodiments, administration of apolynucleotide, pharmaceutical composition, or formulation describedherein to a subject with chikungunya fever reduces the duration of atleast one symptom of the disease in the subject, e.g., reduces theduration of fever, polyarthralgia, and/or arthritis in a subject. Insome embodiments, administration of a polynucleotide, pharmaceuticalcomposition, or formulation described herein to a subject withchikungunya fever reduces the duration of at least one symptom of thedisease in the subject, e.g., reduces the duration of fever,polyarthralgia, and/or arthritis in a subject. In some embodiments,administration of a polynucleotide, pharmaceutical composition, orformulation described herein to a subject with chikungunya fever reducesthe duration of at least one symptom of the disease in the subject by 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, or30 or more days. In some embodiments, administration of apolynucleotide, pharmaceutical composition, or formulation describedherein to a subject with chikungunya fever reduces the duration of atleast one symptom of the disease in the subject by at least 6 hours, atleast 12 hours, at least 24 hours, at least 36 hours, at least 48 hours,at least 72 hours, at least 96 hours, at least 168 hours, at least 336hours, or at least 720 hours or more.

One aspect of the invention provides a method of protecting a humansubject from chikungunya virus infection after the subject has beenexposed to chikungunya virus. In some embodiments, the administration ofthe polynucleotide, pharmaceutical composition or formulation of theinvention protects the human subject from chikungunya virus for at least24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 336 hours, or 720hours or more. In some embodiments, the administration of a single doseof a polynucleotide, pharmaceutical composition or formulation describedherein protects the human subject from chikungunya virus for at least 24hours, 48 hours, 72 hours, 96 hours, 168 hours, 336 hours, or 720 hoursor more.

One aspect of the invention provides a method of protecting a humansubject from the onset of chikungunya fever after the subject has beenexposed to chikungunya virus. In some embodiments, the administration ofthe polynucleotide, pharmaceutical composition or formulation of theinvention protects the human subject from the onset of chikungunya feverfor at least 24 hours, 48 hours, 72 hours, 96 hours, 168 hours, 336hours, or 720 hours or more. In some embodiments, the administration ofa single dose of a polynucleotide, pharmaceutical composition orformulation described herein protects the human subject from the onsetof chikungunya fever for at least 24 hours, 48 hours, 72 hours, 96hours, 168 hours, 336 hours, or 720 hours or more.

One aspect of the invention provides a method of systematicallyproducing an anti-chikungunya virus antibody (anti-CHIKV antibody) in ahuman subject at a level of at least 5 μg/ml, 10 μg/ml, 15 μg/ml, 20μg/ml, 25 μg/ml, or 30 μg/ml for at least 24 hours, 48 hours, 72 hours,96 hours, 168 hours, 336 hours, 720 hours or more after a single doseadministration of a polynucleotide, pharmaceutical composition orformulation described herein.

In some embodiments, the administration of the polynucleotide,pharmaceutical composition or formulation of the invention results inexpression of an anti-CHIKV antibody, or functional portion thereof, incells of the subject. For example, in some embodiments, thepolynucleotides of the present invention are used in methods ofadministering a composition or formulation comprising an mRNA encodingat least one anti-CHIKV antibody polypeptide to a subject, wherein themethod results expression of at least one anti-CHIKV antibodypolypeptide in at least some cells of a subject.

In some embodiments, the administration of the polynucleotide,pharmaceutical composition or formulation of the invention results inexpression of an anti-CHIKV antibody, or functional portion thereof, inat least some of the cells of a subject that persists for a period oftime sufficient to allow some neutralization of CHIKV to occur, e.g.,neutralization of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of CHIKV in the cells.

In some embodiments, the method or use comprises administering at leastone polynucleotide, e.g., mRNA, comprising a nucleotide sequence havingsequence similarity to a polynucleotide selected from SEQ ID NO:2 andSEQ ID NO:4, or at least one polynucleotide selected from SEQ ID NO:5and SEQ ID NO:6, wherein the polynucleotide encodes an anti-CHIKVantibody polypeptide. In some embodiments, the method of use comprisesadministering two polynucleotides, e.g., mRNAs, wherein the twopolynucleotides have nucleotide sequences having sequence similarity toSEQ ID NOs: 2 and 4, respectively, or the two polynucleotides are SEQ IDNOs: 5 and 6.

Other aspects of the present disclosure relate to transplantation ofcells containing polynucleotides to a mammalian subject. Administrationof cells to mammalian subjects is known to those of ordinary skill inthe art, and includes, but is not limited to, local implantation (e.g.,topical or subcutaneous administration), organ delivery or systemicinjection (e.g., intravenous injection or inhalation), and theformulation of cells in pharmaceutically acceptable carriers.

In some embodiments, the polynucleotides (e.g., mRNA), pharmaceuticalcompositions and formulations used in the methods of the inventioncomprise a uracil-modified sequence encoding an anti-CHIKV antibodypolypeptide disclosed herein and a miRNA binding site disclosed herein,e.g., a miRNA binding site that binds to miR-142 and/or a miRNA bindingsite that binds to miR-126. In some embodiments, the uracil-modifiedsequence encoding an anti-CHIKV antibody polypeptide comprises at leastone chemically modified nucleobase, e.g., N1 methylpseudouracil or5-methoxyuracil. In some embodiments, at least 95% of a type ofnucleobase (e.g., uracil) in a uracil-modified sequence encoding ananti-CHIKV antibody polypeptide of the invention are modifiednucleobases. In some embodiments, at least 95% of uracil in auracil-modified sequence encoding an anti-CHIKV antibody polypeptide is1-N-methylpseudouridine or 5-methoxyuridine. In some embodiments, thepolynucleotide (e.g., a RNA, e.g., a mRNA) disclosed herein isformulated with a delivery agent comprising, e.g., a compound having theFormula (I), e.g., any of Compounds 1-232, e.g., Compound II; a compoundhaving the Formula (III), (IV), (V), or (VI), e.g., any of Compounds233-342, e.g., Compound VI; or a compound having the Formula (VIII),e.g., any of Compounds 419-428, e.g., Compound I, or any combinationthereof. In some embodiments, the delivery agent comprises Compound II,DSPC, Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio ofabout 47.5:10.5:39.0:3.0 or about 50:10:38.5:1.5 or about 50:10:38:2. Insome embodiments, the delivery agent comprises Compound VI, DSPC,Cholesterol, and Compound I or PEG-DMG, e.g., with a mole ratio in therange of about 30 to about 60 mol % Compound II or VI (or relatedsuitable amino lipid) (e.g., 30-40, 40-45, 45-50, 50-55 or 55-60 mol %Compound II or VI (or related suitable amino lipid)), about 5 to about20 mol % phospholipid (or related suitable phospholipid or “helperlipid”) (e.g., 5-10, 10-15, or 15-20 mol % phospholipid (or relatedsuitable phospholipid or “helper lipid”)), about 20 to about 50 mol %cholesterol (or related sterol or “non-cationic” lipid) (e.g., about20-30, 30-35, 35-40, 40-45, or 45-50 mol % cholesterol (or relatedsterol or “non-cationic” lipid)) and about 0.05 to about 10 mol % PEGlipid (or other suitable PEG lipid) (e.g., 0.05-1, 1-2, 2-3, 3-4, 4-5,5-7, or 7-10 mol % PEG lipid (or other suitable PEG lipid)). Anexemplary delivery agent can comprise mole ratios of, for example,47.5:10.5:39.0:3.0 or 50:10:38.5:1.5. In certain instances, an exemplarydelivery agent can comprise mole ratios of, for example,47.5:10.5:39.0:3; 47.5:10:39.5:3; 47.5:11:39.5:2; 47.5:10.5:39.5:2.5;47.5:11:39:2.5; 48.5:10:38.5:3; 48.5:10.5:39:2; 48.5:10.5:38.5:2.5;48.5:10.5:39.5:1.5; 48.5:10.5:38.0:3; 47:10.5:39.5:3; 47:10:40.5:2.5;47:11:40:2; 47:10.5:39.5:3; 48:10.5:38.5:3; 48:10:39.5:2.5; 48:11:39:2;or 48:10.5:38.5:3. In some embodiments, the delivery agent comprisesCompound II or VI, DSPC, Cholesterol, and Compound I or PEG-DMG, e.g.,with a mole ratio of about 47.5:10.5:39.0:3.0. In some embodiments, thedelivery agent comprises Compound II or VI, DSPC, Cholesterol, andCompound I or PEG-DMG, e.g., with a mole ratio of about47.5:10.5:39.0:3.0 or about 50:10:38.5:1.5. In some embodiments, thedelivery agent comprises Compound II, DSPC, Cholesterol, and Compound Iwith a mole ratio of about 50:10:38:2. In some embodiments, the deliveryagent comprises Compound II, DSPC, Cholesterol, and Compound I orPEG-DMG, e.g., with a mole ratio in the range of about 30 to about 60mol % Compound II (or related suitable amino lipid) (e.g., 30-40, 40-45,45-50, 50-55 or 55-60 mol % Compound II (or related suitable aminolipid)), about 5 to about 20 mol % phospholipid (or related suitablephospholipid or “helper lipid”) (e.g., 5-10, 10-15, or 15-20 mol %phospholipid (or related suitable phospholipid or “helper lipid”)),about 20 to about 50 mol % cholesterol (or related sterol or“non-cationic” lipid) (e.g., about 20-30, 30-35, 35-40, 40-45, or 45-50mol % cholesterol (or related sterol or “non-cationic” lipid)) and about0.05 to about 10 mol % PEG lipid (or other suitable PEG lipid) (e.g.,0.05-1, 1-2, 2-3, 3-4, 4-5, 5-7, or 7-10 mol % PEG lipid (or othersuitable PEG lipid)).

The skilled artisan will appreciate that the therapeutic effectivenessof a drug or a treatment of the instant invention can be characterizedor determined by measuring the level of expression of an encoded protein(e.g., antibody polypeptide) in a sample or in samples taken from asubject (e.g., from a preclinical test subject (rodent, primate, etc.)or from a clinical subject (human). Likewise, the therapeuticeffectiveness of a drug or a treatment of the instant invention can becharacterized or determined by measuring the level of activity of anencoded protein (e.g., antibody polypeptide) in a sample or in samplestaken from a subject (e.g., from a preclinical test subject (rodent,primate, etc.) or from a clinical subject (human). Furthermore, thetherapeutic effectiveness of a drug or a treatment of the instantinvention can be characterized or determined by measuring the level ofvirus in sample(s) taken from a subject. Levels of protein and/or viruscan be determined post-administration with a single dose of an mRNAtherapeutic of the invention or can be determined and/or monitored atseveral time points following administration with a single dose or canbe determined and/or monitored throughout a course of treatment, e.g., amulti-dose treatment.

The polynucleotides, pharmaceutical compositions, and formulationsdescribed herein may be administered by any route which results in atherapeutically effective outcome. These include, but are not limited,to intradermal, intramuscular, and/or subcutaneous administration. Thepresent disclosure provides methods comprising administering RNAtreatments to a subject in need thereof. The exact amount required willvary from subject to subject, depending on the species, age, and generalcondition of the subject, the severity of the disease, the particularcomposition, its mode of administration, its mode of activity, and thelike. mRNA compositions are typically formulated in dosage unit form forease of administration and uniformity of dosage. It will be understood,however, that the total daily usage of mRNA compositions may be decidedby the attending physician within the scope of sound medical judgment.The specific therapeutically effective, prophylactically effective, orappropriate imaging dose level for any particular patient will dependupon a variety of factors including the disorder being treated and theseverity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the patient; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific compound employed; and like factors wellknown in the medical arts.

In some embodiments, mRNA compositions may be administered at dosagelevels sufficient to deliver 0.0001 mg/kg to 100 mg/kg, 0.001 mg/kg to0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kg to 0.005 mg/kg, 0.05mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1 mg/kg to 40 mg/kg, 0.5mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1 mg/kg to 10 mg/kg, or 1mg/kg to 25 mg/kg, of subject body weight per day, one or more times aday, per week, per month, etc. to obtain the desired therapeutic,diagnostic, prophylactic, or imaging effect (see e.g., the range of unitdoses described in International Publication No WO2013078199, hereinincorporated by reference in its entirety). The desired dosage may bedelivered three times a day, two times a day, once a day, every otherday, every third day, every week, every two weeks, every three weeks,every four weeks, every 2 months, every three months, every 6 months,etc. In certain embodiments, the desired dosage may be delivered usingmultiple administrations (e.g., two, three, four, five, six, seven,eight, nine, ten, eleven, twelve, thirteen, fourteen, or moreadministrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. In exemplaryembodiments, mRNA compositions may be administered at dosage levelssufficient to deliver 0.0005 mg/kg to 0.01 mg/kg, e.g., about 0.0005mg/kg to about 0.0075 mg/kg, e.g., about 0.0005 mg/kg, about 0.001mg/kg, about 0.002 mg/kg, about 0.003 mg/kg, about 0.004 mg/kg or about0.005 mg/kg.

In some embodiments, mRNA compositions may be administered once or twice(or more) at dosage levels sufficient to deliver 0.025 mg/kg to 0.250mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025 mg/kg to 0.750 mg/kg, or 0.025mg/kg to 1.0 mg/kg.

In some embodiments, mRNA compositions may be administered twice (e.g.,Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28,Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 monthslater, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0and 10 years later) at a total dose of or at dosage levels sufficient todeliver a total dose of 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975mg, or 1.0 mg. Higher and lower dosages and frequency of administrationare encompassed by the present disclosure. For example, an RNA treatmentcomposition may be administered three or four times.

In some embodiments, mRNA compositions may be administered twice (e.g.,Day 0 and Day 7, Day 0 and Day 14, Day 0 and Day 21, Day 0 and Day 28,Day 0 and Day 60, Day 0 and Day 90, Day 0 and Day 120, Day 0 and Day150, Day 0 and Day 180, Day 0 and 3 months later, Day 0 and 6 monthslater, Day 0 and 9 months later, Day 0 and 12 months later, Day 0 and 18months later, Day 0 and 2 years later, Day 0 and 5 years later, or Day 0and 10 years later) at a total dose of or at dosage levels sufficient todeliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg or 0.400 mg.

In some embodiments, the RNA for use in a method of treating a subjectis administered to the subject in a single dosage of between 10 μg/kgand 400 μg/kg of the nucleic acid treatment in an effective amount totreat the subject. In some embodiments, the RNA for use in a method oftreating a subject is administered to the subject in a single dosage ofbetween 10 μg and 400 μg of the nucleic acid treatment in an effectiveamount to treat the subject.

An RNA pharmaceutical composition described herein can be formulatedinto a dosage form described herein, such as an intranasal,intratracheal, or injectable (e.g., intravenous, intraocular,intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal,and subcutaneous).

This disclosure is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The disclosure iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting.

Construct Sequences

mRNA ORF Sequence ORF Sequence 5′ UTR 3′ UTR Construct Name (Amino Acid)(Nucleotide) Sequence Sequence Sequence SEQ ID NO: 1 2 13 14 5 ChikV24METDTLLLWVLLLW AUGGAAACCGACAC GGGAAA UGAUAA SEQ ID heavy VPGSTGQVQLVESGGACUGCUGCUGUGGG UAAGAG UAGGCU NO: 5 chain GVVQPGKSLRLSCAA UGCUGCUUCUUUGGAGAAAA GGAGCC consists SGFTFRNYGMHWVR GUGCCCGGAUCUAC GAAGAG UCGGUGfrom 5′ to QAPGKGLDWVALIS AGGACAGGUGCAGC UAAGAA GCCUAG 3′ end: 5′YDGTHKYYKDSLKG UGGUUGAAUCUGGC GAAAUA CUUCUU UTR of RFTISRDNFQNTVDLGGCGGAGUUGUGCA UAAGAC GCCCCU SEQ ID QINSLRPDDTAVYYC GCCUGGCAAGUCUCCCCGGC UGGGCC NO: 13, AKELATSGVVEPLDS UGAGACUGAGCUGU GCCGCC UCCCCC ORFWGQGTLVTVSSAST GCCGCCAGCGGCUU ACC CAGCCC sequence KGPSVFPLAPSSKSTSCACCUUCAGAAACU CUCCUC of SEQ ID GGTAALGCLVKDYF ACGGCAUGCACUGG CCCUUCNO: 2, and PEPVTVSWNSGALTS GUCCGACAGGCUCC CUGCAC 3′ UTR ofGVHTFPAVLQSSGLY AGGCAAAGGCCUUG CCGUAC SEQ ID SLSSVVTVPSSSLGTQAUUGGGUCGCCCUG CCCCGU NO: 14 TYICNVNHKPSNTKV AUCAGCUACGACGG GGUCUUDKKVEPKSCDKTHTC CACCCACAAGUACU UGAAUA PPCPAPELLGGPSVFL ACAAGGACAGCCUGAAGUCU FPPKPKDTLMISRTPE AAGGGCAGAUUCAC GAGUGG VTCVVVDVSHEDPECAUCAGCCGGGACA GCGGC VKFNWYVDGVEVHN ACUUCCAGAACACC AKTKPREEQYNSTYRGUGGACCUGCAGAU VVSVLTVLHQDWLN CAACAGCCUGAGGC GKEYKCKVSNKALPCUGACGACACCGCC APIEKTISKAKGQPRE GUGUACUACUGCGC PQVYTLPPSRDELTK CAAAGAGCUGGCUA NQVSLTCLVKGFYPS  CAAGCGGCGUGGUG DIAVEWESNGQPENN GAACCUCUGGAUUC YKTTPPVLDSDGSFF  UUGGGGACAGGGCA LYSKLTVDKSRWQQCCCUGGUCACAGUG GNVFSCSVLHEALHS  UCUAGCGCCUCUAC HYTQKSLSLSPGKAAAGGGACCCAGCG UGUUCCCUCUGGCU CCUAGCAGCAAGAG CACAAGCGGAGGAACAGCCGCUCUGGGC UGUCUGGUCAAGGA CUACUUUCCCGAGC CUGUGACCGUGUCCUGGAAUUCUGGCGC UCUGACAUCCGGCG UGCACACCUUUCCA GCUGUGCUGCAAAGCAGCGGCCUGUACU CUCUGAGCAGCGUC GUGACAGUGCCAAG CAGCUCUCUGGGCACCCAGACCUACAUC UGCAACGUGAACCA CAAGCCUAGCAACA CCAAGGUGGACAAGAAGGUGGAACCCAA GAGCUGCGACAAGA CCCACACCUGUCCA CCCUGUCCUGCUCCAGAACUGCUCGGCG GACCUUCCGUGUUC CUGUUUCCUCCAAA GCCUAAGGACACCCUGAUGAUCAGCAGA ACACCCGAAGUGAC CUGCGUGGUGGUGG ACGUGUCUCACGAGGACCCUGAAGUGAA GUUCAAUUGGUACG UGGACGGCGUGGAA GUGCACAACGCCAAGACCAAGCCUAGAG AGGAACAGUACAAC AGCACCUACAGAGU GGUGUCCGUGCUGACCGUGCUGCACCAG GAUUGGCUGAACGG CAAAGAGUACAAGU GCAAGGUGUCCAACAAGGCCCUGCCUGC UCCUAUCGAGAAGA CCAUCAGCAAGGCC AAGGGCCAGCCUAGGGAACCUCAGGUGU ACACACUGCCUCCA AGCAGGGACGAGCU GACCAAGAAUCAGGUGUCCCUGACCUGC CUCGUGAAGGGCUU CUACCCUUCCGAUA UCGCCGUGGAGUGGGAGAGCAACGGCCA GCCUGAGAACAACU ACAAGACCACUCCU CCUGUGCUGGACAGCGACGGCUCAUUCU UCCUGUACAGCAAG CUGACAGUGGACAA GUCCAGGUGGCAGCAGGGCAACGUGUUC AGCUGCAGCGUGCU GCACGAAGCCCUGC ACAGCCACUACACCCAGAAGUCCCUGUC UCUGAGCCCUGGCA AA ChikV24 heavy chain portionsSignal sequence Amino acids 1-20 of Nucleotides 1-60 of SEQ ID NO: 1SEQ ID NO: 2 Variable region (VH) Amino acids 21-142 ofNucleotides 61-426 of SEQ ID NO: 1 SEQ ID NO: 2 HCDR1Amino acids 46-53 of Nucleotides 136-159 of SEQ ID NO: 1 SEQ ID NO: 2(underlined) (underlined) HCDR2 Amino acids 71-78 ofNucleotides 211-234 of SEQ ID NO: 1 SEQ ID NO: 2 (underlined)(underlined) HCDR3 Amino acids 117-131 of Nucleotides 349-393 ofSEQ ID NO: 1 SEQ ID NO: 2 (underlined) (underlined) Constant regionAmino acids 143-472 of Nucleotides 427-1416 of SEQ ID NO: 1 SEQ ID NO: 2Chemistry: G5 - all uracils (U) in the mRNA are N1-methylpseudouracilsCap: C1 PolyA tail: 100 nt mRNA ORF Sequence ORF Sequence 5′ UTR 3′ UTRConstruct Name (Amino Acid) (Nucleotide) Sequence Sequence SequenceSEQ ID NO: 3 4 13 14 6 ChikV24 METPAQLLFLLLLWL AUGGAAACACCCGC GGGAAAUGAUAA SEQ ID light chain PDTTGEIVLTQSPGTL UCAGCUGCUGUUCC UAAGAG UAGGCUNO: 6 SLSPGERATLSCRAS UGCUGCUGCUGUGG AGAAAA GGAGCC consistsQSLVSSYFGWYQQK CUGCCUGAUACCAC GAAGAG UCGGUG from 5′ to RGQSPRLLIYAASTRAGGCGAGAUCGUGC UAAGAA GCCUAG 3′ end: 5′ ATGIPDRFSGSGSGTD UGACACAGAGCCCUGAAAUA CUUCUU UTR of FTLTISRLEPEDFAVY GGCACACUGUCACU UAAGAC GCCCCUSEQ ID YCQQYGNTPFTFGGG GUCUCCAGGCGAAA CCCGGC UGGGCC NO: 13,TKVEIKRTVAAPSVFI GAGCCACACUGAGC GCCGCC UCCCCC ORF FPPSDEQLKSGTASVUGUAGAGCCAGCCA ACC CAGCCC sequence VCLLNNFYPREAKVQ GAGCCUGGUGUCCA CUCCUCof SEQ ID WKVDNALQSGNSQE GCUACUUCGGCUGG CCCUUC NO: 4, andSVTEQDSKDSTYSLS UAUCAGCAGAAGAG CUGCAC 3′ UTR of STLTLSKADYEKHKVAGGCCAGUCUCCUC CCGUAC SEQ ID YACEVTHQGLSSPVT GGCUGCUGAUCUAC CCCCGUNO: 14 KSFNRGEC GCCGCUUCUACAAG GGUCUU AGCCACCGGCAUUC UGAAUACCGAUAGAUUCAGC AAGUCU GGCUCUGGCAGCGG GAGUGG CACCGAUUUCACCC GCGGCUGACAAUCAGCAGA CUGGAACCCGAGGA CUUCGCCGUGUACU ACUGUCAGCAGUACGGCAACACACCCUU CACCUUUGGCGGAG GCACCAAGGUGGAA AUCAAGAGAACAGUGGCUGCUCCCAGCG UGUUCAUCUUCCCA CCUUCCGACGAGCA GCUGAAGUCUGGCACAGCCUCUGUCGUG UGCCUGCUGAACAA CUUCUACCCUCGGG AAGCCAAGGUGCAGUGGAAGGUGGACAA CGCCCUGCAGAGCG GCAACAGCCAAGAG AGCGUGACAGAGCAGGACAGCAAGGACU CCACCUACAGCCUG AGCAGCACACUGAC CCUGAGCAAGGCCGACUACGAGAAGCAC AAGGUGUACGCCUG CGAAGUGACACACC AGGGCCUGUCUAGCCCUGUGACCAAGAG CUUCAACAGAGGCG AGUGC ChikV24 light chain portionsSignal sequence Amino acids 1-20 of Nucleotides 1-60 of SEQ ID NO: 3SEQ ID NO: 4 Variable region (VL) Amino acids 21-128 ofNucleotides 61-384 of SEQ ID NO: 3 SEQ ID NO: 4 LCDR1Amino acids 47-53 of Nucleotides 139-159 of SEQ ID NO: 3 SEQ ID NO: 4(underlined) (underlined) LCDR2 Amino acids 71-73 ofNucleotides 211-219 of SEQ ID NO: 3 SEQ ID NO: 4 (underlined)(underlined) LCDR3 Amino acids 110-118 of Nucleotides 328-354 ofSEQ ID NO: 3 SEQ ID NO: 4 (underlined) (underlined) Constant regionAmino acids 129-235 of Nucleotides 385-705 of SEQ ID NO: 3 SEQ ID NO: 4Chemistry: G5 - all uracils (U) in the mRNA are N1-methylpseudouracilsCap: C1 PolyA tail: 100 nt

EXAMPLES Example 1: Chimeric Polynucleotide Synthesis

A. Triphosphate Route

Two regions or parts of a chimeric polynucleotide can be joined orligated using triphosphate chemistry. According to this method, a firstregion or part of 100 nucleotides or less can be chemically synthesizedwith a 5′ monophosphate and terminal 3′desOH or blocked OH. If theregion is longer than 80 nucleotides, it can be synthesized as twostrands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus canfollow. Monophosphate protecting groups can be selected from any ofthose known in the art.

The second region or part of the chimeric polynucleotide can besynthesized using either chemical synthesis or IVT methods. IVT methodscan include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 80 nucleotides can be chemicallysynthesized and coupled to the IVT region or part.

It is noted that for ligation methods, ligation with DNA T4 ligase,followed by treatment with DNAse should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then such region or part can comprise a phosphate-sugarbackbone.

Ligation can then be performed using any known click chemistry,orthoclick chemistry, solulink, or other bioconjugate chemistries knownto those in the art.

B. Synthetic Route

The chimeric polynucleotide can be made using a series of startingsegments. Such segments include:

-   -   (a) Capped and protected 5′ segment comprising a normal 3′OH        (SEG. 1)    -   (b) 5′ triphosphate segment which can include the coding region        of a polypeptide and comprising a normal 3′OH (SEG. 2)    -   (c) 5′ monophosphate segment for the 3′ end of the chimeric        polynucleotide (e.g., the tail) comprising cordycepin or no 3′OH        (SEG. 3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) can be treatedwith cordycepin and then with pyrophosphatase to create the5′monophosphate.

Segment 2 (SEG. 2) can then be ligated to SEG. 3 using RNA ligase. Theligated polynucleotide can then be purified and treated withpyrophosphatase to cleave the diphosphate. The treated SEG. 2-SEG. 3construct is then purified and SEG. 1 is ligated to the 5′ terminus. Afurther purification step of the chimeric polynucleotide can beperformed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments can be represented as: 5′ UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′ UTR+PolyA (SEG. 3).

The yields of each step can be as much as 90-95%.

Example 2: PCR for cDNA Production

PCR procedures for the preparation of cDNA can be performed using 2×KAPAHIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, MA). This systemincludes 2×KAPA ReadyMix12.5 μl; Forward Primer (10 μM) 0.75 μl; ReversePrimer (10 μM) 0.75 μl; Template cDNA—100 ng; and dH₂O diluted to 25.0μl. The PCR reaction conditions can be: at 95° C. for 5 min. and 25cycles of 98° C. for 20 sec, then 58° C. for 15 sec, then 72° C. for 45sec, then 72° C. for 5 min. then 4° C. to termination.

The reverse primer of the instant invention can incorporate a poly-T120for a poly-A120 in the mRNA. Other reverse primers with longer orshorter poly(T) tracts can be used to adjust the length of the poly(A)tail in the polynucleotide mRNA.

The reaction can be cleaned up using Invitrogen's PURELINK™ PCR MicroKit (Carlsbad, CA) per manufacturer's instructions (up to 5 μg). Largerreactions will require a cleanup using a product with a larger capacity.Following the cleanup, the cDNA can be quantified using the NANODROP™and analyzed by agarose gel electrophoresis to confirm the cDNA is theexpected size. The cDNA can then be submitted for sequencing analysisbefore proceeding to the in vitro transcription reaction.

Example 3: In Vitro Transcription (IVT)

The in vitro transcription reactions can generate polynucleotidescontaining uniformly modified polynucleotides. Such uniformly modifiedpolynucleotides can comprise a region or part of the polynucleotides ofthe invention. The input nucleotide triphosphate (NTP) mix can be madeusing natural and un-natural NTPs.

A typical in vitro transcription reaction can include the following:

-   -   1 Template cDNA—1.0 μg    -   2 10× transcription buffer (400 mM Tris-HCl pH 8.0, 190 mM        MgCl₂, 50 mM DTT, 10 mM Spermidine)—2.0 μl    -   3 Custom NTPs (25 mM each)—7.2 μl    -   4 RNase Inhibitor—20 U    -   5 T7 RNA polymerase—3000 U    -   6 dH₂O—Up to 20.0 μl. and    -   7 Incubation at 37° C. for 3 hr-5 hrs.

The crude IVT mix can be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase can then be used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA can bepurified using Ambion's MEGACLEAR™ Kit (Austin, TX) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA can be quantified using the NanoDrop andanalyzed by agarose gel electrophoresis to confirm the RNA is the propersize and that no degradation of the RNA has occurred.

Example 4: Enzymatic Capping

Capping of a polynucleotide can be performed with a mixture includes:IVT RNA 60 μg-180 μg and dH₂O up to 72 μl. The mixture can be incubatedat 65° C. for 5 minutes to denature RNA, and then can be transferredimmediately to ice.

The protocol can then involve the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂O (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The polynucleotide can then be purified using Ambion's MEGACLEAR™ Kit(Austin, TX) following the manufacturer's instructions. Following thecleanup, the RNA can be quantified using the NANODROP™ (ThermoFisher,Waltham, MA) and analyzed by agarose gel electrophoresis to confirm theRNA is the proper size and that no degradation of the RNA has occurred.The RNA product can also be sequenced by running areverse-transcription-PCR to generate the cDNA for sequencing.

Example 5: PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This can be done by mixingCapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂O up to 123.5 μl and incubating at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction can be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, TX) (up to 500 μg). Poly-A Polymeraseis, in some cases, a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction does not always result in an exact size polyA tail.Hence polyA tails of approximately between 40-200 nucleotides, e.g.,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe invention.

Example 6: Natural 5′ Caps and 5′ Cap Analogues

5′-capping of polynucleotides can be completed concomitantly during thein vitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, MA). 5′-capping of modified RNA can becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, MA). Cap 1 structure can be generated using both Vaccinia VirusCapping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure can be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure can be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes can be derived from a recombinant source.

When transfected into mammalian cells, the modified mRNAs can have astability of between 12-18 hours or more than 18 hours, e.g., 24, 36,48, 60, 72 or greater than 72 hours.

Example 7: Capping Assays

A. Protein Expression Assay

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be transfected into cells at equal concentrations.After 6, 12, 24 and 36 hours post-transfection, the amount of proteinsecreted into the culture medium can be assayed by ELISA. Syntheticpolynucleotides that secrete higher levels of protein into the mediumwould correspond to a synthetic polynucleotide with a highertranslationally-competent Cap structure.

B. Purity Analysis Synthesis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be compared for purity using denaturing Agarose-Ureagel electrophoresis or HPLC analysis. Polynucleotides with a single,consolidated band by electrophoresis correspond to the higher purityproduct compared to polynucleotides with multiple bands or streakingbands. Synthetic polynucleotides with a single HPLC peak would alsocorrespond to a higher purity product. The capping reaction with ahigher efficiency would provide a more pure polynucleotide population.

C. Cytokine Analysis

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be transfected into cells at multiple concentrations.After 6, 12, 24 and 36 hours post-transfection the amount ofpro-inflammatory cytokines such as TNF-alpha and IFN-beta secreted intothe culture medium can be assayed by ELISA. Polynucleotides resulting inthe secretion of higher levels of pro-inflammatory cytokines into themedium would correspond to polynucleotides containing animmune-activating cap structure.

D. Capping Reaction Efficiency

Polynucleotides encoding a polypeptide, containing any of the capstaught herein, can be analyzed for capping reaction efficiency by LC-MSafter nuclease treatment. Nuclease treatment of capped polynucleotideswould yield a mixture of free nucleotides and the capped5′-5-triphosphate cap structure detectable by LC-MS. The amount ofcapped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and would correspond to cappingreaction efficiency. The cap structure with higher capping reactionefficiency would have a higher amount of capped product by LC-MS.

Example 8: Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) can be loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, CA) and run for12-15 minutes according to the manufacturer protocol.

Example 9: Nanodrop Modified RNA Quantification and UV Spectral Data

Modified polynucleotides in TE buffer (1 μl) can be used for Nanodrop UVabsorbance readings to quantitate the yield of each polynucleotide froma chemical synthesis or in vitro transcription reaction.

Example 10: Formulation of Modified mRNA Using Lipidoids

Polynucleotides can be formulated for in vitro experiments by mixing thepolynucleotides with the lipidoid at a set ratio prior to addition tocells. In vivo formulation can require the addition of extra ingredientsto facilitate circulation throughout the body. To test the ability ofthese lipidoids to form particles suitable for in vivo work, a standardformulation process used for siRNA-lipidoid formulations can be used asa starting point. After formation of the particle, polynucleotide can beadded and allowed to integrate with the complex. The encapsulationefficiency can be determined using a standard dye exclusion assays.

Example 11: Protection Studies in Mice

Mice studies were conducted in accordance with the approval of theInstitutional Animal Care and Use Committee of Utah State University(Protocol #2339). The work was performed in the AAALAC-accreditedLaboratory Animal Research Center of Utah State University (PHSAssurance no. A3801-01) in accordance with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals (Revision;2010).

Male and female AG129 mice, bred in an in-house colony at Utah StateUniversity, were used for protection studies. Animals were assignedrandomly to experimental groups and individually marked with ear tags.The CHIKV-LR-2006 stock was prepared by passaging the virus twice inC6/36 Aedes albopictus cells. The CHIKV stock had a titer of 109-5TCID₅₀/mL. The CHIKV-specific monoclonal antibody, CHIKV24 was collectedfrom hybridoma supernatants and purified by protein G chromatography,and the antibody suspension was supplied in a ready-to-treat liquidform. Virus titers in sera were assayed using an infectious cell cultureassay where a specific volume of serum was added to the first tube of aseries of dilution tubes. Serial dilutions were made and added to Verocell culture monolayers. Three days later cytopathic effect (CPE) wasused to identify the end-point of infection. Four replicates were usedto calculate the TCID₅₀ per mL of serum.

Cages of mice were assigned randomly to groups of 5 animals. Groups ofmice were treated with the ChikV24 antibody via a single IV injection 24h prior to virus challenge (Example 12). Alternatively, similar groupsof animals were given mRNA encoding human antibodies by the IV route(Example 15). Mice then were anesthetized with isoflurane prior tosubcutaneous injection in the footpad and hock of the right leg with1015 TCID₅₀ of CHIKV in a total volume of 0.1 mL (0.05 mL each site).Survival was monitored twice daily through the critical period ofdisease to 7 days post-infection. Serum was collected by cheek veinbleed on day 2 post-infection to measure viremia.

Example 12: Multiple-Dose Study of Anti-Chikungunya Virus Antibody inMice

A multiple-dose study was conducted to determine to what extent anantibody against chikungunya virus can protect mice from the disease.AG129 mice lack receptors for interferon-α/β and -γ and are highlyvulnerable to infection with CHIKV and thus this is a highly stringentmodel for testing antiviral compounds or the protective efficacy ofCHIKV24 antibody (Couderc et al., PLoS Pathog 4, e29 (2008); Kaur andChu, Drug Discov Today, (2013); Partidos et al., Vaccine 29, 3067-3073(2011); and Wang et al., Journal of virology 85, 9249-9252(2011)). AG129mice were intravenously injected with a single 10 mg/kg, 2 mg/kg, or 0.4mg/kg dose of the CHIKV24 antibody, an anti-CHIKV antibody, or theCR9114 anti-influenza antibody as a negative control, via tail vein IVbolus. Five mice were tested at each dose. Mice were challenged 24 hourslater with a lethal dose of CHIKV (chikungunya virus strain LR06(LR2006-OPY1, 2C6) strain at a dose of 10^(2.5) TCID₅₀) by inoculationof the footpad and hock of the right leg (total volume of 0.1 mL of thediluted virus (0.05 mL each site)). Animals were monitored daily formorbidity (e.g., by measuring weight loss) and mortality for up to 21days after challenge. Naïve mice that were not injected with antibody orchallenged with virus were also used as a control. Mice injected withantibody were bled at 24-hours post-injection to measure total human IgG(huIgG) concentration. Protein was detected using a total human IgGELISAkit (Abcam, ab100547).

FIG. 1A shows that there was a dose-dependent concentration of human IgGin mouse serum. Mice that had received 10 mg/kg (200 μg), 2 mg/kg (40μg) or 0.4 mg/kg (8 μg) of recombinant CHIKV24 IgG protein had meansystemic CHIKV24 IgG concentrations of 78 μg/mL, 10 μg/mL or 3 μg/mL,respectively. The serum concentration of the influenza control antibodywas similar to that of CHIKV24. (n=5 at each dose).

FIG. 1B shows that 100% of the mice that had received a prior infusionof either a 10 mg/kg dose or a 2 mg/kg dose of the CHIKV24 antibodysurvived for 21 days following challenge with virus. Intermediatesurvival was observed after treatment with 0.4 mg/kg of the antibody, as50% of the mice injected with the CHIKV24 antibody survived for 21 daysfollowing challenge with virus. By contrast, all of the control miceinjected with an anti-influenza antibody died by 5 days followingchallenge with chikungunya virus. All unchallenged control animalssurvived. A comparison of the survival results with the achievedconcentration levels of serum human IgG (FIG. 1A) indicated that theCHIKV24 IgG could protect AG129 mice in a lethal challenge model atsystemic levels of 10 μg/mL of antibody at the time of challenge.

Viruses

Virus suspensions of CHIKV attenuated vaccine strain 181/25 were grownon Vero cell monolayer cultures, and supernatant was harvested 36 hourspost-inoculation and clarified by centrifugation at 2,000×rpm for 10 minat 4° C. The CHIKV East/Central/South African [ECSA] genotype strainused for neutralization screening in this study was SL15649 (accessionnumber GU189061). For in vivo studies, the Reunion Island CHIKV isolateLR2006-OPYI was obtained. Stocks for these viruses were prepared inC6/36 Aedes albopictus cells.

Example 13: Synthesis of mRNA Encoding Human Anti-Chikungunya Antibody

Sequence optimized polynucleotides encoding human anti-chikungunyaantibody heavy chain polypeptides, i.e., SEQ ID NO:1, and light chainpolypeptides, i.e., SEQ ID NO:3, are synthesized and characterized asdescribed in Examples 1 to 11, and prepared for the Examples describedbelow.

An mRNA encoding human anti-chikungunya antibody heavy chain polypeptidecan be constructed, e.g., by using an ORF sequence provided in SEQ IDNO:2. An mRNA encoding human anti-chikungunya antibody light chainpolypeptide can be constructed, e.g., by using an ORF sequence providedin SEQ ID NO:4. The mRNA sequence includes both 5′ and 3′ UTR regionsflanking the ORF sequence (nucleotide). In an exemplary construct, the5′ UTR and 3′ UTR sequences are SEQ ID NO:13 and SEQ ID NO:14,respectively (see Sequence Listing).

5′UTR: (SEQ ID NO: 13)GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGC CGCCACC 3′UTR:(SEQ ID NO: 14) UGAUAAUAGGCUGGAGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAA UAAAGUCUGAGUGGGCGGC

The antibody heavy and light chain mRNA sequences are prepared asmodified mRNA. Specifically, during in vitro translation, modified mRNAcan be generated using N1-methylpseudouridine-5′-Triphosphate or5-methoxy-UTP to ensure that the mRNAs contain 100%N1-methylpseudouridine-5′-Triphosphate or 5-methoxy-uridine instead ofuridine. Further, mRNA can be synthesized with a primer that introducesa polyA-tail, and a Cap 1 structure is generated on both mRNAs usingVaccinia Virus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl.

Example 14: Production and Characterization of Nanoparticle Compositions

A. Production of Nanoparticle Compositions

Nanoparticles can be made with mixing processes such as microfluidicsand T-junction mixing of two fluid streams, one of which contains thepolynucleotide and the other has the lipid components.

Lipid compositions are prepared by combining an ionizable amino lipiddisclosed herein, e.g., a lipid according to Formula (I) such asCompound II or a lipid according to Formula (III) such as Compound VI, aphospholipid (such as DOPE or DSPC, obtainable from Avanti Polar Lipids,Alabaster, AL), a PEG lipid (such as 1,2-dimyristoyl-sn-glycerolmethoxypolyethylene glycol, also known as PEG-DMG, obtainable fromAvanti Polar Lipids, Alabaster, AL), and a structural lipid (such ascholesterol, obtainable from Sigma-Aldrich, Taufkirchen, Germany, or acorticosteroid (such as prednisolone, dexamethasone, prednisone, andhydrocortisone), or a combination thereof) at concentrations of about 50mM in ethanol. Solutions should be refrigerated for storage at, forexample, −20° C. Lipids are combined to yield desired molar ratios anddiluted with water and ethanol to a final lipid concentration of betweenabout 5.5 mM and about 25 mM.

Nanoparticle compositions including a polynucleotide and a lipidcomposition are prepared by combining the lipid solution with a solutionincluding the a polynucleotide at lipid composition to polynucleotidewt:wt ratios between about 5:1 and about 50:1. The lipid solution israpidly injected using a NanoAssemblr microfluidic based system at flowrates between about 10 ml/min and about 18 ml/min into thepolynucleotide solution to produce a suspension with a water to ethanolratio between about 1:1 and about 4:1.

For nanoparticle compositions including an RNA, solutions of the RNA atconcentrations of 0.1 mg/ml in deionized water are diluted in 50 mMsodium citrate buffer at a pH between 3 and 4 to form a stock solution.

Nanoparticle compositions can be processed by dialysis to remove ethanoland achieve buffer exchange. Formulations are dialyzed twice againstphosphate buffered saline (PBS), pH 7.4, at volumes 200 times that ofthe primary product using Slide-A-Lyzer cassettes (Thermo FisherScientific Inc., Rockford, IL) with a molecular weight cutoff of 10 kD.The first dialysis is carried out at room temperature for 3 hours. Theformulations are then dialyzed overnight at 4° C. The resultingnanoparticle suspension is filtered through 0.2 μm sterile filters(Sarstedt, Nümbrecht, Germany) into glass vials and sealed with crimpclosures. Nanoparticle composition solutions of 0.01 mg/ml to 0.10 mg/mlare generally obtained.

The method described above induces nano-precipitation and particleformation. Alternative processes including, but not limited to,T-junction and direct injection, can be used to achieve the samenano-precipitation.

B. Characterization of Nanoparticle Compositions

A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire,UK) can be used to determine the particle size, the polydispersity index(PDI) and the zeta potential of the nanoparticle compositions in 1×PBSin determining particle size and 15 mM PBS in determining zetapotential.

Ultraviolet-visible spectroscopy can be used to determine theconcentration of a polynucleotide (e.g., RNA) in nanoparticlecompositions. 100 μL of the diluted formulation in 1×PBS is added to 900μL of a 4:1 (v/v) mixture of methanol and chloroform. After mixing, theabsorbance spectrum of the solution is recorded, for example, between230 nm and 330 nm on a DU 800 spectrophotometer (Beckman Coulter,Beckman Coulter, Inc., Brea, CA). The concentration of polynucleotide inthe nanoparticle composition can be calculated based on the extinctioncoefficient of the polynucleotide used in the composition and on thedifference between the absorbance at a wavelength of, for example, 260nm and the baseline value at a wavelength of, for example, 330 nm.

For nanoparticle compositions including an RNA, a QUANT-IT™ RIBOGREEN®RNA assay (Invitrogen Corporation Carlsbad, CA) can be used to evaluatethe encapsulation of an RNA by the nanoparticle composition. The samplesare diluted to a concentration of approximately 5 μg/mL in a TE buffersolution (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the dilutedsamples are transferred to a polystyrene 96 well plate and either 50 μLof TE buffer or 50 μL of a 2% Triton X-100 solution is added to thewells. The plate is incubated at a temperature of 37° C. for 15 minutes.The RIBOGREEN® reagent is diluted 1:100 in TE buffer, and 100 μL of thissolution is added to each well. The fluorescence intensity can bemeasured using a fluorescence plate reader (Wallac Victor 1420Multilablel Counter; Perkin Elmer, Waltham, MA) at an excitationwavelength of, for example, about 480 nm and an emission wavelength of,for example, about 520 nm. The fluorescence values of the reagent blankare subtracted from that of each of the samples and the percentage offree RNA is determined by dividing the fluorescence intensity of theintact sample (without addition of Triton X-100) by the fluorescencevalue of the disrupted sample (caused by the addition of Triton X-100).

Exemplary formulations of the nanoparticle compositions are presented inTable 6 below. The term “Compound” refers to an ionizable lipid such asMC3, Compound II, or Compound VI. “Phospholipid” can be DSPC or DOPE.“PEG-lipid” can be PEG-DMG or Compound I.

TABLE 6 Exemplary Formulations of Nanoparticles Composition (mol %)Components 40:20:38.5:1.5 Compound:Phospholipid:Chol:PEG-lipid45:15:38.5:1.5 Compound:Phospholipid:Chol:PEG-lipid 50:10:38.5:1.5Compound:Phospholipid:Chol:PEG-lipid 55:5:38.5:1.5Compound:Phospholipid:Chol:PEG-lipid 60:5:33.5:1.5Compound:Phospholipid:Chol:PEG-lipid 45:20:33.5:1.5Compound:Phospholipid:Chol:PEG-lipid 50:20:28.5:1.5Compound:Phospholipid:Chol:PEG-lipid 55:20:23.5:1.5Compound:Phospholipid:Chol:PEG-lipid 60:20:18.5:1.5Compound:Phospholipid:Chol:PEG-lipid 40:15:43.5:1.5Compound:Phospholipid:Chol:PEG-lipid 50:15:33.5:1.5Compound:Phospholipid:Chol:PEG-lipid 55:15:28.5:1.5Compound:Phospholipid:Chol:PEG-lipid 60:15:23.5:1.5Compound:Phospholipid:Chol:PEG-lipid 40:10:48.5:1.5Compound:Phospholipid:Chol:PEG-lipid 45:10:43.5:1.5Compound:Phospholipid:Chol:PEG-lipid 55:10:33.5:1.5Compound:Phospholipid:Chol:PEG-lipid 60:10:28.5:1.5Compound:Phospholipid:Chol:PEG-lipid 40:5:53.5:1.5Compound:Phospholipid:Chol:PEG-lipid 45:5:48.5:1.5Compound:Phospholipid:Chol:PEG-lipid 50:5:43.5:1.5Compound:Phospholipid:Chol:PEG-lipid 40:20:40:0Compound:Phospholipid:Chol:PEG-lipid 45:20:35:0Compound:Phospholipid:Chol:PEG-lipid 50:20:30:0Compound:Phospholipid:Chol:PEG-lipid 55:20:25:0Compound:Phospholipid:Chol:PEG-lipid 60:20:20:0Compound:Phospholipid:Chol:PEG-lipid 40:15:45:0Compound:Phospholipid:Chol:PEG-lipid 45:15:40:0Compound:Phospholipid:Chol:PEG-lipid 50:15:35:0Compound:Phospholipid:Chol:PEG-lipid 55:15:30:0Compound:Phospholipid:Chol:PEG-lipid 60:15:25:0Compound:Phospholipid:Chol:PEG-lipid 40:10:50:0Compound:Phospholipid:Chol:PEG-lipid 45:10:45:0Compound:Phospholipid:Chol:PEG-lipid 50:10:40:0Compound:Phospholipid:Chol:PEG-lipid 55:10:35:0Compound:Phospholipid:Chol:PEG-lipid 60:10:30:0Compound:Phospholipid:Chol:PEG-lipid 50:10:38:2Compound:Phospholipid:Chol:PEG-lipid

Example 15: Multiple-Dose Study of In Vivo Expression of mRNA EncodingAnti-Chikungunya Antibody in Mice, and Protection Against Lethal VirusChallenge

To assess the ability of mRNAs encoding the human heavy and light chainsof the ChikV24 antibody to facilitate protein expression in vivo, mRNAsencoding the heavy and light chains were co-formulated at a 2:1 heavychain:light chain (HC:LC) w/w ratio, and intravenously administered intoAG129 mice via tail vein IV bolus at 0.5 mg/kg, 0.1 mg/kg, or 0.02 mg/kgof each mRNA. Five mice were tested at each dose. The mRNA wasformulated in Compound II- and PEG-DMG-containing lipid nanoparticles(LNPs) for delivery into the mice and stored at 4° C. until use. Micewere challenged 24 hours later with Chikungunya virus strain LR06(LR2006-OPY1, 2C6) at a dose of 10^(2.5) TCID50 by footpad inoculation.Animals were monitored daily for morbidity (e.g., by measuring weightloss) and mortality for up to 21 days after challenge. Control mice wereinjected with mRNA encoding an antibody that does not bind tochikungunya virus (the CR9114 anti-influenza antibody, as a controlantibody). An additional group of animals was infused with the test mRNAdoses at the same time and were bled at 24-hours, 48-hours, and 72-hourspost-injection to measure total human IgG (huIgG) concentration (in theabsence of virus challenge after infusion). Protein was detected using atotal human IgG ELISA kit (Abcam, ab100547).

The virus titers in the tissues and serum of test and control mice wereassayed using an infectious cell culture assay where a specific volumeof either tissue homogenate or plasma was added to the first tube of aseries of dilution tubes. Serial dilutions were made and added to Verocell monolayer cultures two days after virus challenge to determinevirus titer (log₁₀ TCID₅₀/mL). Three days later cytopathic effect (CPE)was used to identify the end-point of infection. Four replicates wereused to calculate the 50% tissue culture infectious doses (TCID₅₀) permL of plasma or gram of tissues.

FIG. 2A shows that infusion of mice with the mRNAs resulted in theexpression of human ChikV24 antibody in vivo. There was a dose-dependenteffect, as the highest serum concentrations of human IgG at 24 hourspost-injection were observed in mice that were injected with 0.5 mg/kgof the mRNAs. The mean peak serum concentration of the 0.5 mg/kg treatedgroup was 14.9 μg/mL. Each group had 5 animals.

FIG. 2B shows that a dose-responsive improvement in survival of AG129mice infected with chikungunya virus was observed after treatment withChikV24 mRNA administered intravenously 24 hours prior to viruschallenge as a prophylaxis (**P<0.01, as compared with placebo). 100% ofthe mice that were administered 0.5 mg/kg of the mRNAs encoding theheavy and light chains of the ChikV24 antibody (top line, amounting to aserum concentration of approximately 10 μg/mL), and 40% of the mice thatwere administered 0.1 mg/kg of the mRNAs encoding the ChikV24 antibody(middle line, amounting to a serum concentration of 3 μg/mL) survivedfor 21 days following challenge with virus. Mice that were administered0.02 mg/kg of mRNAs encoding the heavy and light chains of ChikV24(bottom line, amounting to 0.5 μg/mL) did not survive. Despite the lowerlevel of protection at the two lower doses of mRNAs (0.1 mg/kg and 0.02mg/kg), the survival curves for mice that received these doses wereimproved (P<0.01), showing delayed mortality, compared to mice thatreceived placebo treatment (mRNA encoding an irrelevant IgG that doesnot bind chikungunya virus), demonstrating a benefit of the CHIKV24 mRNAtreatment even at the lower doses tested. Thus, the mRNA-encoded ChikV24antibody has potency at equivalent levels as the corresponding purifiedrecombinant antibody. The number of animals in each group was 10.

A comparison of the serum levels of human IgG achieved by mRNA infusionmeasured in a parallel group of non-challenged animals receiving 0.5mg/kg or 0.1 mg/kg of IgG (see FIG. 2A) with the results of the survivalexperiments (FIG. 2B) indicated that the CHIKV24 mRNA treatment couldcompletely protect AG129 mice in the lethal challenge model when a 10μg/mL concentration of systemic ChikV24 antibody was achieved, while atleast half of the virus challenged animals were protected at ChikV24antibody serum levels of about 3 μg/mL.

FIG. 2C shows that mRNA-expressed ChikV24 antibody significantly reducedchikungunya virus titers below the level of detection in the serum ofAG129 mice at 2 days following virus challenge at all mRNA doses (0.5mg/kg, 0.1 mg/kg, and 0.02 mg/kg) relative to control mice that wereintravenously administered 0.5 mg/kg of mRNA encoding an antibody thatdoes not bind to chikungunya virus (***P<0.0003 (Kruskal Wallis testwith Dunn's post test), as compared to the control IgG). The limit ofdetection (LOD) was 1.7. Control mice exhibited an average of 4.6 log₁₀50% tissue culture infectious doses (TCID₅₀). Although virus was notobserved in the serum in the low-dose treatment group (0.02 mg/kg),virus likely replicated in other tissues, since mortality occurred. Thereduction of viremia to the limit of detection corroborated atherapeutic effect against viral replication. The number of animals ineach group was 5.

Example 16: Pharmacokinetics Arm to Multiple-Dose Study of In VivoExpression of mRNA Encoding Anti-Chikungunya Antibody in Mice

To study the pharmacokinetics of the human ChikV24 antibody expressedfrom modified mRNAs encoding the light and heavy chains of the antibody,AG129 mice were intravenously injected via tail vein IV bolus with 0.5mg/kg, 0.1 mg/kg, or 0.02 mg/kg of each mRNA. Five mice were injected ateach dose, and control mice were administered PBS. mRNAs were formulatedin Compound II- and PEG-DMG containing lipid nanoparticles prior toadministration. None of the mice were challenged with chikungunya virusafter mRNA injection. Mice were bled prior to injection, and at24-hours, 48-hours, and 72-hours post-injection to measure total humanIgG (huIgG) concentration. Protein was detected using a total human IgGELISA kit (Abcam, ab100547).

The results of the pharmacokinetics analysis are provided in Table 7.

TABLE 7 In vivo duration of IgG Expression over 72 hours Group μg/mLHuman IgG dose 0 hr 24 hr 48 hr 72 hr  0.5 mpk 0.01 18.5 10.7 11.6 0.013.4 3 2.3 0.01 13.9 9.5 7.1 0.01 14.1 10.1 7.4 0.01 0.01 0.01 0.01  0.1mpk 0.01 3.7 3.4 2 0.01 5.9 5 2.9 0.01 2.3 2.1 2 0.01 1.4 1.3 1.2 0.015.3 6 2.5 0.02 mpk 0.01 0.01 0.01 0.01 0.01 0.3 0.3 0.2 0.01 0.9 1.1 0.60.01 0.6 0.8 0.4 0.01 0.5 0.8 0.5 control 0.01 0.01 0.01 0.01 0.01 0.010.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 LOQ: 0.01 μg/mL

As shown in FIG. 3 and in Table 7, administration of increasing amountsof mRNA encoding the ChikV24 antibody resulted in greater amounts ofantibody in the serum of animals at 24, 48, and 72-hours post-injection.One animal in the 0.5 mg/kg dose group and one animal in the 0.02 mg/kgdose group failed to respond to mRNA injection, i.e., failed to expressantibody from injected mRNAs.

Example 17: Single-Dose Study of mRNA Encoding Anti-Chikungunya Antibodyin Mice and Protection Against Arthritis and Musculoskeletal Disease

While immunocompromised mice provide a stringent protection model formRNA encoding human anti-ChikV antibody, chikungunya virus infection israrely fatal in humans but instead causes severe, acute and chronicpolyarthralgia and polyarthritis. Accordingly, mRNA encoding ChikV24antibody was tested to see if it could reduce prevent or reduce symptomsin an immunocompetent mouse model of CHIKV-induced arthritis andmusculoskeletal disease by administering the mRNA after virus exposure.In this mouse model, subcutaneous virus infection results in a biphasicswelling of the infected foot peaking at 3 and 7 days post-inoculation(dpi). Four-week-old wild-type C57BL/6J mice (000664; JacksonLaboratories) were inoculated subcutaneously in the left footpad with10³ FFU of CHIKV-LR in Hank's Balanced Salt Solution (HBSS) supplemented1% heat-inactivated (HI)-FBS. mRNAs encoding the human heavy and lightchains of the ChikV24 antibody (co-formulated at a 2:1 heavy chain:lightchain (HC:LC) w/w ratio) were intravenously injected via tail vein IVbolus at 10 mg/kg of mRNA into C57BL/6 mice at 4 hours after the micewere challenged with Chikungunya virus strain LR06 (LR2006-OPY1, 2C6) ata lethal dose of 10^(2.5) TCID50 by footpad inoculation. The mRNA wasformulated in Compound II- and PEG-DMG-containing lipid nanoparticles(LNPs) prior to delivery into the mice. Control mice were injected withmRNA encoding an antibody that does not bind to chikungunya virus (theCR9114 anti-influenza antibody). Ipsilateral foot swelling in the micewas monitored via measurements (width×height) using digital calipers(n=15/group, two experiments, two-way ANOVA with Sidak's post-test).Serum was collected at 2 dpi, and mice were sacrificed and perfusedextensively with 20 mL of PBS at 7 dpi and ipsilateral (i.) andcontralateral (c.) ankles were harvested. Serum and tissues were titeredfor chikungunya virus RNA by qRT-PCR using RNA isolated from viralstocks as a standard curve to determine FFU equivalents, as previouslydescribed (Fox et al., Broadly Neutralizing Alphavirus Antibodies Bindan Epitope on E2 and Inhibit Entry and Egress. Cell 163, 1095-1107(2015), herein incorporated by reference in its entirety). Viral RNA wasquantified by qRT-PCR in the serum (n=15/group, two experiments, forserum n=10/group, two experiments, Mann Whitney test for each tissue,for ankles). For histology, the ipsilateral feet collected at 7 dpi werefixed in 4% paraformaldehyde (PFA) for 24 hours, rinsed with PBS andwater, and decalcified for 14 days in 14% EDTA free acid (Sigma) at pH7.2. The decalcified tissue was then rinsed, dehydrated, embedded inparaffin, sectioned, and stained with hematoxylin and eosin (H & E).Images were acquired on a Nikon Eclipse E400 microscope.

FIG. 4A shows that wild-type (WT) C57BL/6 mice injected with mRNAsencoding human ChikV24 antibody at 4 hours after virus challenge did notdevelop foot swelling compared to the control mice that received an mRNALNP encoding an irrelevant IgG control antibody.

FIG. 4B shows that chikungunya virus titers were very low (at the limitof detection) in the serum collected from most of the mice injected withmRNAs encoding ChikV24 antibody at 2 dpi. By contrast, high levels ofviremia were observed in the control mice injected with mRNA encoding anantibody that does not bind to chikungunya virus. FIG. 4C shows thatipsilateral ankles collected from mice injected with mRNAs encodingChikV24 antibody at 7 dpi had an 80-fold reduction in viral RNA, with nospread to the contralateral ankle, compared to the ipsilateral anklescollected from the control mice.

FIG. 4D shows the results of the histological analysis of theipsilateral feet collected from mice injected with mRNAs encodingChikV24 antibody at 7 dpi compared to the ipsilateral feet collectedfrom control mice that received an mRNA LNP encoding an irrelevant IgGcontrol antibody. The top panels of FIG. 4D show large cellularinfiltration of chikungunya virus into the joint space of the controlmRNA treated mice (top left panel), whereas cellular infiltration wasabsent in the mice injected with ChikV24-encoding mRNA (top middle andright panels). The histological results also showed compared thecellular infiltration of chikungunya virus in the midfoot of test miceversus control mice (bottom panels of FIG. 4D). Slides from two of fivemice administered mRNAs encoding ChikV24 antibody exhibited minimalcellular infiltration of virus in the midfoot (bottom right panel),although three of the test mice exhibited detectable cellularinfiltration in the soft tissue (bottom middle panel). However, theextent of immune cells and edema in the midfoot of mice injected withChikV24-encoding mRNAs was reduced markedly compared to the midfoot ofcontrol mice (bottom left panel). These results show that mRNAs encodinghuman ChikV24 antibody confer protection in an immunocompetent mousemodel of arthritis caused by chikungunya virus infection.

Example 18: Housing of Nonhuman Primates for Studies

Nonhuman primate studies were conducted at Charles River Laboratories(Sherbrooke, Quebec, Canada). Animal experiments and husbandry followedNIH guidelines (NIH Publications No. 8023, eighth edition) and the USANational Research Council and the Canadian Council on Animal Care (CCAC)guidelines. No treatment randomization or blinding methods were used forany of the animal studies. Sample sizes were determined by the resourceequation method. The repeat-dose NHP study was conducted under GLPconditions.

Macaques used for study were 2 to 3 years old males and weighed between2.3 and 2.8 kg at the initiation of dosing. Tuberculin tests werecarried out on arrival at the test facility and were negative. Animalswere housed socially (up to 3 animals of same sex and same dosing grouptogether) in stainless steel cages equipped with a stainless-steel meshfloor and an automatic watering valve, with the exception of times whenthey were separated for designated study procedures/activities. Animalswere housed in a temperature- and humidity-controlled environment(21-26° C. and 30-70%, respectively), with an automatic 12-hourdark/light cycle. Primary enclosures were as specified in the USDAAnimal Welfare Act (9 CFR, Parts 1, 2 and 3) and as described in theGuide for the Care and Use of Laboratory Animals (39). PMI NutritionInternational Certified Primate Chow No. 5048 (25% protein) was providedtwice daily, except during designated procedures. The chow was providedin amounts appropriate for the size and age of the animals. Municipaltap water after treatment by reverse osmosis and ultraviolet irradiationwas made freely available to each animal via an automatic wateringsystem (except during designated procedures).

Example 19: Single Dose Study of mRNA-Expressed Chikungunya VirusAntibody in Cynomolgus Monkeys

To test the expression levels of human anti-ChikV antibody from modifiedmRNAs in a nonhuman primate, mRNAs encoding the heavy and light chainsof ChikV24 were delivered intravenously into cynomolgus macaques. Onegoal of the experiment was to determine whether the CHIKV24 mRNAs couldinduce expression of human IgG in the serum of monkeys at levels thatcorrespond to the protective serum concentrations observed in mice. 0.5mg/kg of the mRNAs encoding the heavy and light antibody chains wereco-formulated in Compound II- and Compound I-containing lipidnanoparticles prior to administration. The mRNAs encoding the ChikV24antibody were infused over 60 minutes in a volume of 5 mL/kg and a doseconcentration of 0.02 mg/mL. A total of 6 monkeys were injected with asingle dose of mRNAs encoding the ChikV24 antibody (Study 1). This studywas repeated with 6 macaques per group (Study 2). The followingparameters and end points were also evaluated in this study: clinicalsigns, body weights, food evaluation, and human IgG expression in serum.

The concentration and duration of human antibody was assayed in serumcollected from injected monkeys at all time points. Blood samples (0.3mL) were collected in serum separator tubes on day 1 (at pre-dose and 6,24, 96, 168, 336, or 720 hours after the start of infusion) and on day82. The blood samples were maintained at ambient temperature for atarget of 30 min following collection, then processed to serum within 90min of collection. The samples were centrifuged for 10 min in arefrigerated centrifuge (set to maintain 4° C.) at 1,200×g. Theresulting serum was separated, aliquoted, and frozen immediately overdry ice before storage at −80° C. Human IgG in serum was analyzed usingan ELISA a Human Therapeutic IgG1 ELISA Kit (Cayman Chemical, #500910).The kit instructions were followed exactly with serum dilutions rangingfrom 1:100 to 1:1,000. A standard curve of absorbance at 450 nm versuslog (concentration) was fit with a 4-parameter logistic equation forIgG1 quantification. Human IgG pharmacokinetic parameters were estimatedusing Phoenix software (Certara, USA) using a non-compartmental approach(NCA), consistent with the intravenous route of administration.Parameters were estimated using nominal sampling times relative to thestart of each dose administration. Concentration values reported asBelow Quantifiable Limit were assigned a value of zero. The area underthe concentration vs. time curve (AUC) was calculated using the lineartrapezoidal method with linear interpolation. AUC values were reportedto 3 significant digits, and t_(1/2) values were reported to one decimalplace. The terminal elimination phase for each subject was estimatedusing at least three observed concentration values. The slope of theelimination phase was determined using log linear regression on theunweighted concentration data. As shown in FIG. 5A, the ChikV24 antibodywas expressed from modified mRNAs injected in monkeys over the course of720-hours post-injection. There were no test article-related clinicalsigns, changes in body weight, or changes in food consumption during thecourse of this study. IgG1 expression peaked at 24 hours after the startof infusion for animals that received a 0.5 mg/kg dose of mRNAs encodingthe ChikV24 antibody. Table 8 shows that the mean human IgG levels at 24hours post-injection was 10.1 to 35.9 μg/mL (a maximum concentration of35.9 μg/mL in Study 1 and 10.1 μg/mL in Study 2). The differences inpeak expression level across the two studies can be attributed to assayand study variability. The half-life of the mRNA-expressed ChikV24antibody was 23 days in cynomolgus macaques. Thus, the mRNA infusionsachieved protective concentrations of the ChikV24 antibody in macaques.

TABLE 8 Human IgG pharmacokinetic parameters of ChikV24 antibody inmacaques following delivery of modified mRNAs encoding the antibodyAUC_(0-720 hr) T_(max) (hr) C_(max) (μg/mL) (hr * μg/mL) t_(1/2) (hr)Mean SD CV % Mean SD CV % Mean SD CV % Mean SD CV% 24 0 0 10.1 5.36 533,720 1,950 52.4 561 65.8 11.7

Next, the function of the ChikV24 antibodies expressed in serum from theinjected modified mRNAs was compared to the function of the recombinantChikV24 monoclonal antibody. The serum samples (from Study 2) at the24-hour timepoint from the pharmacodynamics studies (FIG. 5A) weretested for the presence of CHIKV-specific binding or neutralizingantibodies. Antibody function was assessed by a 50% focus reductionneutralization test (FRNT₅₀) and ELISA. A group of 6 animals was tested(Study 2), and in vitro experiments were conducted twice. A standardcurve for concentration versus activity in each assay was generatedusing dilution curves of purified recombinant ChikV24 antibody atdefined concentrations. FIG. 5B shows that the functional equivalents ofChikV24 antibody activity measured using the FRNT₅₀ and ELISA methods,by comparison to the activity of the sera with the ChikV24 antibodystandard curves, were within the variability of the assays, suggestingthat the mRNA-expressed antibody was fully functional.

Example 20: Multiple Dose Study of mRNA-Expressed Chikungunya VirusAntibody in Cynomolgus Monkeys

To test the expression of human anti-ChikV antibody over time innon-human primates from multiple doses of modified mRNAs, two doses ofmRNAs encoding the heavy and light chains of the ChikV24 antibody weredelivered intravenously into cynomolgus monkeys one week apart (on days0 and 7). Animals were administered mRNA doses of 0.3 mg/kg, 1 mg/kg, or3.0 mg/kg, or a PBS control. The mRNAs were co-formulated in CompoundII- and Compound I-containing lipid nanoparticles prior toadministration. Necropsy was then performed on study animals on day 8(following the second injection of mRNAs), or on day 98 after a 12-weektreatment-free recovery period. Multiple serum samples were collectedthroughout the duration of the study to measure the concentrations ofthe ChikV24 antibody in serum after multiple mRNA doses. Serum wascollected at 6, 24, 48, 72 and 120 hours after the start of infusion ofdose 1 and at 6, 12, 24, 48, 72, 120, 168, 216, 288, 360, 432, 528, 720,1,080 and 2,160 hours after the start of infusion of dose 2. Antibodyconcentrations after day 8 were calculated only for the highest mRNAdose level (3 mg/kg).

FIG. 6 shows that the ChikV24 antibody was detected in the serum samplesof cynomolgus monkeys injected with multiple doses of mRNAs encoding theantibody. A dose-dependent response was observed, as ChikV24 IgG serumconcentrations were higher with increasing doses of mRNAs. MaximumChikV24 IgG serum concentrations of 16.2 μg/mL and 28.8 μg/mL wereobserved in animals administered the high dose of 3.0 mg/kg mRNA at 24hours (day 1) following the first dose and 24 hours (day 8) followingthe second dose, respectively. Sex-based differences were not detectedin ChikV24 IgG serum levels. ChikV24 IgG serum levels were detectedthrough 100 days after the second dose (at 3.0 mg/kg, administered onday 7) in animals that had a recovery period, with an average serumconcentration of 2.9 μg/mL. Human IgG antibodies were detectable throughday 83 when dosed once at 0.5 mg/kg.

Example 21: Administration of mRNAs Encoding Chikungunya Virus Antibodyin Humans

mRNA constructs encoding the heavy and light chains (SEQ ID NO:5 and SEQID NO:6, respectively) of the human ChikV24 antibody are formulated inlipid nanoparticles (LNPs) containing Compound II, DSPC, Cholesterol,and Compound I (at a molar ratio of 50:10:38:2) and are administeredintravenously to humans who have been infected with, or are at risk ofbeing infected with, chikungunya virus. Administering the mRNAs encodingthe ChikV24 antibody is expected to reduce symptoms associated withchikungunya virus infection in individuals who have been exposed to thevirus. Prophylactically administering the mRNAs encoding the ChikV24antibody to individuals at greater risk of being exposed to chikungunyavirus is expected to prevent infection and/or reduce disease symptomsshould infection occur.

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03. It should be appreciatedthat embodiments described in this document using an open-endedtransitional phrase (e.g., “comprising”) are also contemplated, inalternative embodiments, as “consisting of” and “consisting essentiallyof” the feature described by the open-ended transitional phrase. Forexample, if the disclosure describes “a composition comprising A and B”,the disclosure also contemplates the alternative embodiments “acomposition consisting of A and B” and “a composition consistingessentially of A and B”.

What is claimed is:
 1. A polynucleotide comprising an mRNA comprising:(i) a 5′ UTR; (ii) an open reading frame (ORF) encoding a polypeptidecomprising the heavy chain variable region of the heavy chain antibodysequence of SEQ ID NO:1, wherein the ORF comprises a nucleic acidsequence that is at least 80% identical to nucleotides 61-426 of SEQ IDNO:2; (iii) a stop codon; and (iv) a 3′ UTR.
 2. A polynucleotidecomprising an mRNA comprising: (i) a 5′ UTR; (ii) an open reading frame(ORF) encoding a polypeptide comprising the light chain variable regionof the light chain antibody sequence of SEQ ID NO:3, wherein the ORFcomprises a nucleic acid sequence that is at least 80% identical tonucleotides 61-384 of SEQ ID NO:4; (iii) a stop codon; and (iv) a 3′UTR.
 3. A pharmaceutical composition comprising the polynucleotide ofclaim 1, and a delivery agent.
 4. A pharmaceutical compositioncomprising: a first polynucleotide comprising a first mRNA comprising(i) a first 5′ UTR, (ii) a first open reading frame (ORF) encoding afirst polypeptide comprising the heavy chain variable region of theheavy chain antibody sequence of SEQ ID NO:1, wherein the first ORFcomprises a first nucleic acid sequence that is at least 80% identicalto nucleotides 61-426 of SEQ ID NO:2, (iii) a first stop codon, and (iv)a first 3′ UTR; a second polynucleotide comprising a second mRNAcomprising (i) a second 5′ UTR, (ii) a second ORF encoding a secondpolypeptide comprising the light chain variable region of the lightchain antibody sequence of SEQ ID NO:3, wherein the second ORF comprisesa second nucleic acid sequence that is at least 80% identical tonucleotides 61-384 of SEQ ID NO:4, (iii) a second stop codon, and (iv) asecond 3′ UTR; and a delivery agent, wherein the first polypeptide whenpaired with the second polypeptide forms an anti-Chikungunya virusantibody or an anti-Chikungunya virus antibody fragment.
 5. Thepharmaceutical composition of claim 4, wherein the first nucleic acidsequence is at least 90% identical to SEQ ID NO:2, and wherein thesecond nucleic acid sequence is at least 90% identical to SEQ ID NO:4.6. The pharmaceutical composition of claim 4, wherein the first mRNA andthe second mRNA each comprise a 5′ terminal cap.
 7. The pharmaceuticalcomposition of claim 6, wherein each 5′ terminal cap comprises a Cap0,Cap0, ARCA, inosine, N1-methyl-guanosine, 2′-fluoro-guanosine,7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine,2-azidoguanosine, Cap2, Cap4, 5′ methylG cap, or an analog thereof. 8.The pharmaceutical composition of claim 4, wherein the first mRNA andthe second mRNA each comprise a poly-A region.
 9. The pharmaceuticalcomposition of claim 4, wherein the first mRNA and the second mRNA eachcomprise at least one chemically modified nucleobase, sugar, backbone,or any combination thereof.
 10. The pharmaceutical composition of claim9 wherein the at least one chemically modified nucleobase is selectedfrom the group consisting of pseudouracil (ψ), N1-methylpseudouracil(m1ψ), 1-ethylpseudouracil, 2-thiouracil (s2U), 4′-thiouracil,5-methylcytosine, 5-methyluracil, 5-methoxyuracil, and any combinationthereof.
 11. The pharmaceutical composition of claim 9, wherein all ofthe uracils of the first mRNA and the second mRNA areN1-methylpseudouracils.
 12. A method of expressing an anti-chikungunyavirus antibody in a human subject in need thereof, comprisingadministering to the human subject an effective amount of thepharmaceutical composition of claim
 4. 13. A method of reducingchikungunya virus levels in a human subject in need thereof, comprisingadministering to the human subject an effective amount of thepharmaceutical composition of claim
 4. 14. The method of claim 12,wherein the pharmaceutical composition is administered to the humansubject multiple times at a frequency of about once a week, about onceevery two weeks, or about once a month.
 15. The method of claim 12,wherein the pharmaceutical composition is administered intravenously.16. The method of claim 12, wherein the pharmaceutical composition isadministered subcutaneously.
 17. The pharmaceutical composition of claim4, wherein the first nucleic acid sequence is at least 80% identical tonucleotides 61-1416 of SEQ ID NO:2, and wherein the second nucleic acidsequence is at least 80% identical to nucleotides 61-705 of SEQ ID NO:4.18. The pharmaceutical composition of claim 4, wherein the first nucleicacid sequence is at least 80% identical to SEQ ID NO:2, and wherein thesecond nucleic acid sequence is at least 80% identical to SEQ ID NO:4.19. The pharmaceutical composition of claim 4, wherein the first nucleicacid sequence is at least 90% identical to nucleotides 61-426 of SEQ IDNO:2, and wherein the second nucleic acid sequence is at least 90%identical to nucleotides 61-384 of SEQ ID NO:4.
 20. The pharmaceuticalcomposition of claim 4, wherein the first nucleic acid sequence is atleast 90% identical to nucleotides 61-1416 of SEQ ID NO:2, and whereinthe second nucleic acid sequence is at least 90% identical tonucleotides 61-705 of SEQ ID NO:4.
 21. The pharmaceutical composition ofclaim 4, wherein the first nucleic acid sequence is 100% identical tonucleotides 61-426 of SEQ ID NO:2, and wherein the second nucleic acidsequence is 100% identical to nucleotides 61-384 of SEQ ID NO:4.
 22. Thepharmaceutical composition of claim 4, wherein the first nucleic acidsequence is 100% identical to nucleotides 61-1416 of SEQ ID NO:2, andwherein the second nucleic acid sequence is 100% identical tonucleotides 61-705 of SEQ ID NO:4.
 23. The pharmaceutical composition ofclaim 4, wherein the first nucleic acid sequence is 100% identical toSEQ ID NO:2, and wherein the second nucleic acid sequence is 100%identical to SEQ ID NO:4.